Biomedical subjects

Y Yoshida

Publications and source records attributed to Y Yoshida.

At least 91 records · Page 5Linked to original sources

PubMed2001

Charge separation in a novel artificial photosynthetic reaction center lives 380 ms.

An extremely long-lived charge-separated state has been achieved successfully using a ferrocene-zincporphyrin-freebaseporphyrin-fullerene tetrad which reveals a cascade of photoinduced energy transfer and multistep electron transfer within a molecule in frozen media as well as in solutions. The lifetime of the resulting charge-separated state (i.e., ferricenium ion-C(60) radical anion pair) in a frozen benzonitrile is determined as 0.38 s, which is more than one order of magnitude longer than any other intramolecular charge recombination processes of synthetic systems, and is comparable to that observed for the bacterial photosynthetic reaction center. Such an extremely long lifetime of the tetrad system has been well correlated with the charge-separated lifetimes of two homologous series of porphyrin-fullerene dyad and triad systems.

Journal Article↗

PubMed2001

Surface molecules essential for positive selection are retained but interfered in thymic epithelial cells after monolayer culture.

It has been reported that the three-dimensional structure of thymic epithelial cells (TECs) is responsible for thymic positive selection but that this ability disappears when TECs are cultured in monolayer. These results have supported the hypothesis that certain TEC-specific molecules are extinguished during monolayer culture. In this study, using MHC class II-restricted T-cell receptor transgenic mice, we demonstrated that preselected CD4(+)8(+) (DP) thymocytes were inhibited from developing into CD4(+)8(-) (CD4SP) cells in reaggregate thymus organ culture with monolayer-cultured TECs, but this inhibition was removed when TECs were cultured in monolayer with protein synthesis inhibitor or when the cultured TECs were treated with fixative. These results seem to be inconsistent with the previous hypothesis and indicate that monolayer culture allows TECs to retain the surface molecules necessary for positive selection but interferes with their function, which must be sustained for three dimensional structure.

Animals↗

PubMed2001

Similar subunit interactions contribute to assembly of clathrin adaptor complexes and COPI complex: analysis using yeast three-hybrid system.

Clathrin adaptor protein (AP) complexes are heterotetramers composed of two large, one medium, and one small subunits. By exploiting the yeast three-hybrid system, we have found that an interaction between the two large subunits of the AP-1 complex, gamma-adaptin and beta1-adaptin, is markedly enhanced in the presence of the small subunit, sigma1. Similarly, two large subunits of the AP-4 complex, epsilon-adaptin and beta4-adaptin, are found to interact with each other only in the presence of the small subunit, sigma4. Furthermore, we have found that an interaction between two large subunits of the COPI F subcomplex, gamma-COP and beta-COP, is detectable only in the presence of zeta-COP. Because these COPI subunits have common ancestral origins to the corresponding AP subunits, these three-hybrid data, taken together with the previous two-hybrid data, suggest that the AP complexes and the COPI F subcomplex assemble by virtue of similar subunit interactions.

Adaptor Protein Complex 1↗

PubMed2001

Changes in CSF hypocretin-1 (orexin A) levels in rats across 24 hours and in response to food deprivation.

Hypocretin-1 is consistently detectable in the CSF of healthy human subjects, but is absent in narcoleptics. However, functional roles of CSF hypocretin are largely unknown. We examined fluctuation of CSF hypocretin-1 across 24 h and in response to food restriction in rats. Hypocretin-1 levels were high during the dark period when animals were active, but decreased by 40% toward the end of the light (rest) period. After 72 h food deprivation hypocretin-1 levels during the rest phase increased to concentrations similar to those seen during the baseline active phase; however, no increase in response to food deprivation was observed during the active phase. These results indicate an important link between circadian control of sleep and energy homeostasis via the hypocretin system.

Animals↗

PubMed2001

Enantioselective total synthesis of a potent antitumor antibiotic, fredericamycin A.

The asymmetric total synthesis of both enantiomers of the potent antitumor antibiotic fredericamycin A (1) is detailed based on the protocol for the construction of its peri-hydroxy polyaromatic skeleton bearing the chirality at the spiro carbon via a strong base-induced cycloaddition of suitably substituted homophthalic anhydrides (AB-ring unit) with an optically active CDEF-ring unit. Particular attention has been given to the novel synthesis of the optically active spiro carbon center by a stereospecific rearrangement of optically active benzofuzed-trans-epoxy acylates leading to spirocyclopentane-1,1'-indane systems. This method is quite useful for the construction of an optically active spiro compound and was applied to the synthesis of the optically pure CDEF-ring unit of 1. Cycloaddition of the optically pure CDEF-ring unit to AB-ring units prepared via benzyne afforded two natural and unnatural-type hexacyclic compounds, which were converted to natural and unnatural enantiomers of synthetic 1, and the absolute configuration of natural 1 was determined as S.

Antibiotics, Antineoplastic↗

PubMed2001

A point mutation in nucleoside diphosphate kinase results in a deficient light response for perithecial polarity in Neurospora crassa.

In Neurospora crassa, the phosphorylation of nucleoside diphosphate kinase (NDK)-1 is rapidly enhanced after blue light irradiation. We have investigated the function of NDK-1 in the blue light signal transduction pathway. A mutant called psp (phosphorylation of small protein) shows undetectable phosphorylation of NDK-1 and is defective in light-responsive regulation of perithecial polarity. Sequencing analysis of ndk-1 cDNA by reverse transcription-polymerase chain reaction revealed that proline 72 of ndk-1 was replaced with histidine in psp. The mutation ndk-1(P72H) resulted in accumulation of normal levels of mRNA and of about 25% of NDK-1(P72H) protein compared with that of wild type as determined by Western blot analysis. The ectopic expression of cDNA and introduction of genomic DNA of wild type ndk-1 in psp (ndk-1(P72H)) suppressed the reduction in accumulation and phosphorylation of NDK-1 and the light-insensitive phenotype. These findings demonstrated that the phenotype of psp was caused by the ndk-1(P72H) mutation. Biochemical analysis using recombinant NDK-1 and NDK-1(P72H) indicated that the P72H substitution in NDK-1 was responsible for the decrease in phosphotransfer activities, 5% of autophosphorylation activity, and 2% of V(max) for protein kinase activity phosphorylating myelin basic protein, compared with those of wild type NDK-1, respectively.

Amino Acid Substitution↗

PubMed2001

Caspase-3 activation by lysosomal enzymes in cytochrome c-independent apoptosis in myelodysplastic syndrome-derived cell line P39.

In most cases, apoptosis is considered to involve mitochondrial dysfunction with sequential release of cytochrome c from mitochondria, resulting in activation of caspase-3. However, we found that etoposide induced apoptosis in P39 cells, a myelodysplastic syndrome-derived cell line, without the release of cytochrome c. Furthermore, in etoposide-treated P39 cells, no changes in mitochondrial membrane potential (delta psi m) were detected by flow cytometry. Flow cytometry using a pH-sensitive probe demonstrated that lysosomal pH increased during early apoptosis in P39 cells treated with etoposide. A reduction in the ATP level preceded the elevation of lysosomal pH. In addition, specific inhibitors of vacuolar H+-ATPase induced apoptosis in P39 cells but not in HL60 cells. Although etoposide-induced activation of caspase-3 was followed by DNA ladder formation in P39 cells, E-64d, an inhibitor of lysosomal thiol proteases, specifically suppressed etoposide-induced activation of caspase-3. Western blotting analysis provided direct evidence for the involvement of a lysosomal enzyme, cathepsin L. These findings indicate that lysosomal dysfunction induced by a reduction in ATP results in leakage of lysosomal enzymes into the cytosolic compartment and that lysosomal enzyme(s) may be involved in activation of caspase-3 during apoptosis in P39 cells treated with etoposide.

Anti-Bacterial Agents↗

PubMed2001

Target gene therapy for alpha-fetoprotein-producing hepatocellular carcinoma by E1B55k-attenuated adenovirus.

Gene therapy using replication-competent adenovirus that selectively propagates in tumor cells may be an effective treatment for cancer. We developed an adenovirus that would be replication specific for hepatocellular carcinoma (HCC). Based on our finding that the E1B55k-deficient adenovirus was able to replicate in human primary hepatocytes, we therefore designed an adenovirus carrying E1A and attenuated E1B gene driven by the alpha-fetoprotein promoter (Adv-AFP-E1AdB), thus restricting the replication specificity in AFP-producing HCC. Replication of Adv-AFP-E1AdB in primary hepatocytes was practically negligible 4 days after infection. Although Adv-AFP-E1AdB replicated slowly in AFP-producing HCC, it efficiently destroyed HCC cells independent of their p53 status. Experiments were conducted in vivo using systemic administration of Adv-AFP-E1AdB and we observed tumor size reduction in nude mice having liver cancer. The use of replication-competent adenovirus deficient of the E1B gene coupled to an AFP-targeting strategy may be a safe and efficacious treatment for HCC.

Adenoviridae↗

PubMed2001

Superconducting gap structure of spin-triplet superconductor Sr2RuO4 studied by thermal conductivity.

To clarify the superconducting gap structure of the spin-triplet superconductor Sr2RuO4, the in-plane thermal conductivity has been measured as a function of relative orientations of the thermal flow, the crystal axes, and a magnetic field rotating within the 2D RuO2 planes. The in-plane variation of the thermal conductivity is incompatible with any model with line nodes vertical to the 2D planes and indicates the existence of horizontal nodes. These results place strong constraints on models that attempt to explain the mechanism of the triplet superconductivity.

Journal Article↗

PubMed2001

Intraventricular administration of the neurotrophic factor midkine ameliorates hippocampal delayed neuronal death following transient forebrain ischemia in gerbils.

Midkine (MK) is a growth factor with neurotrophic activities, and is expressed during the early stages of experimental cerebral infarction in rats in the zone surrounding the infarct. To evaluate in vivo activity of MK in preventing neuronal death, MK produced in yeast (Pichia pastoris) was administered into the brain ventricle immediately before occlusion of the bilateral common carotid artery of Mongolian gerbils. MK administration at the dose of 0.5-2 microg immediately before occlusion was found to ameliorate delayed neuronal death in the hippocampal CA1 region caused by transient ischemia 7 days after the insult. The hippocampal neurons of the MK-administered gerbils tended to degenerate 14 and 21 days after the insult, but their numbers remained higher than those in saline-administered controls; however, the hippocampal neurons were degenerated 28 days after the insult. MK administration at 2 h after occlusion did not ameliorate the neuronal death. These findings suggested that the therapeutic time window was narrow. The two to four times repeated administration of 2 microg MK immediately before and at 1, 2, or 3 weeks after the occlusion were not significantly different for the hippocampal neuronal death at 28 days after the insult compared with a single injection, but were significantly effective compared with vehicle administration alone. These findings suggested that the therapeutic time window was relatively narrow. The potent neuroprotective activity of MK observed in vivo suggested that MK might be useful as a therapeutic reagent for prevention of neuronal death in neurodegenerative diseases.

Animals↗

PubMed2001

Expression of herpes simplex virus-thymidine kinase gene controlled by a promoter region of the midkine gene confers selective cytotoxicity to ganciclovir in human carcinoma cells.

A selective expression of suicide gene(s) in tumor cells should produce a preferential cytotoxic effect on tumors. Promoter region(s) of a gene that is expressed in tumors but not in normal tissues can be useful for tumor-specific transcription of a suicide gene. Midkine (MK), a growth/differentiation factor, is expressed predominantly in various types of human tumors, whereas its expression in adult normal tissues is highly restricted. In our study, we showed that a 2.3-kb fragment of genomic DNA in the 5' upstream region of the MK gene could activate transcription of a fused reporter gene in MK-positive cells but not in MK-negative cells. Efficiency of the cis-acting sequence to permit expression of an exogenous gene in tumor cells was comparable with that of the SV40 promoter. Regulated expression of the herpes simplex virus-thymidine kinase (HSV-TK) gene under the control of the MK promoter conferred increased sensitivity to ganciclovir (GCV) on MK-positive tumor cells. Administration of GCV into nude mice that were implanted with MK-positive tumor cells that expressed the HSV-TK gene under the control of the MK promoter could suppress the subsequent tumor growth. Expression of therapeutic genes restricted to tumors can be achieved by the use of the putative cis-acting MK promoter.

Animals↗

PubMed2001

Y box-binding protein-1 binds preferentially to single-stranded nucleic acids and exhibits 3'-->5' exonuclease activity.

We have previously shown that Y box-binding protein-1 (YB-1) binds preferentially to cisplatin-modified Y box sequences. Based on structural and biochemical data, we predicted that this protein binds single-stranded nucleic acids. In the present study we confirmed the prediction and also discovered some unexpected functional features of YB-1. We found that the cold shock domain of the protein is necessary but not sufficient for double-stranded DNA binding while the C-tail domain interacts with both single-stranded DNA and RNA independently of the cold shock domain. In an in vitro translation system the C-tail domain of the protein inhibited translation but the cold shock domain did not. Both in vitro pull-down and in vivo co-immunoprecipitation assays revealed that YB-1 can form a homodimer. Deletion analysis mapped the C-tail domain of the protein as the region of homodimerization. We also characterized an intrinsic 3'-->5' DNA exonuclease activity of the protein. The region between residues 51 and 205 of its 324-amino acid extent is required for full exonuclease activity. Our findings suggest that YB-1 functions in regulating DNA/RNA transactions and that these actions involve different domains.

Binding Sites↗

PubMed2001

The proteasome inhibitor MG132 promotes accumulation of the steroidogenic acute regulatory protein (StAR) and steroidogenesis.

StAR, a protein synthesized in the cytoplasm and subsequently imported into mitochondria, regulates the rate-determining step in steroidogenesis, the transport of cholesterol from the outer to the inner mitochondrial membrane. The active form of StAR is the 37 kDa pre-protein, which has a short half-life. To determine whether proteasomes participate in the turnover of StAR, we incubated primary cultures of preovulatory rat granulosa cells and immortalized human granulosa cells in the presence of MG132, a specific inhibitor to proteasome catalysis. This treatment caused accumulation of StAR in unstimulated cells. Moreover, incubation of the cells with MG132 in the presence of forskolin (FK), luteinizing hormone/chorionic gonadotropin or follicular stimulating hormone augmented the accumulation of both the 37 kDa cytoplasmic protein and the 30 kDa mature mitochondrial protein, compared to cells incubated with FK or the gonadotropic hormones alone. Concomitantly, progesterone production was enhanced. In contrast no elevation in the 37 kDa StAR intracellular levels or progesterone production was observed following incubation of the cells with the cysteine protease inhibitor E-64. The increase of the 37 kDa StAR protein was evident after 15 min and 30 min of incubation with MG132 (143% and 187% of control values, respectively) with no significant elevation of the 30 kDa protein. Accumulation of the intermediate mitochondrial 32 kDa protein was evident after 1-2 h and the accumulation of the 30 kDa protein was evident only after 4 h of incubation with MG132. In contrast, no elevation in adrenodoxin, a component of the cytochrome P450scc enzyme system, was found. These data suggest that StAR protein is either directly or indirectly degraded by the proteasome which may explain, in part, its short half-life. Moreover, it seems that the cytosolic 37 kDa protein, which is responsible for the steroidogenic activity of StAR, is the primary proteasomal substrate and that the inhibition of its degradation by MG132 causes the up-regulation of progesterone production.

Adrenodoxin↗

PubMed2001

HTLV-1-associated myelopathy/tropical spastic paraparesis with pseudohypoparathyroidism.

In many short-stature patients with human T-lymphotrophic virus type I-associated myelopathy/tropical spastic paraparesis (HAM/TSP), signs and symptoms were manifested during childhood. Successive investigations revealed 12 of 14 short-stature patients with pseudohypoparathyroidism (PHP) from the findings of short metacarpi, parathyroid hormone infusion test, immunoblotting of erythrocyte membrane, or lymphocytic Northern blotting of Gsalpha. Patients with PHP probably showed HAM/TSP based on their modified immunologic status. Human T-lymphotrophic virus type I infection did not induce PHP, but PHP may be a risk factor for the occurrence of HAM/TSP.

Adolescent↗

PubMed2001

Construction of Oka varicella vaccine expressing human immunodeficiency virus env antigen.

Oka varicella vaccine has been used to confer active immunity to varicella-zoster virus (VZV) in healthy and immunocompromised hosts. Based on its attenuated nature, Oka varicella vaccine expressing human immunodeficiency virus (HIV) env antigen was constructed by inserting the HIVenv gene into the viral genome and its immunogenicity was assessed in guinea pigs. The HIVenv gene encoding 296-463 amino acids was inserted between the sequences of the hepatitis B surface antigen and the thymidine kinase gene of the cloned plasmid and the recombinant virus was isolated by cotransfection of the chimeric plasmid with viral DNA. Insertion of the HIVenv gene into the viral genome was confirmed by PCR and sequencing of the viral genome of the recombinant virus. The recombinant virus expressed 30k HIVenv fusion protein in its infected cells. In guinea pigs, immunization with the recombinant virus induced an antibody response to both the HIV antigen and the V3 peptide of gp120 as well as VZV gE:gI. Cell-mediated immunity to the HIV antigen and gE:gI was assessed by the cutaneous reaction representing delayed type hypersensitivity. Immunized guinea pigs responded well to both the HIV antigen and gE:gI. Thus the recombinant Oka varicella vaccine expressing the HIVenv antigen induced both a humoral and cell-mediated immunity to the HIV antigen similar to VZV as Oka varicella vaccine induces humoral and cell-mediated immunity to VZV in the vaccinees. This recombinant Oka varicella vaccine expressing the HIVenv antigen may be evaluated for its immunogenicity as one of the AIDS vaccine candidates.

AIDS Vaccines↗

PubMed2001

Resistance of primary cultured mouse hepatic tumor cells to cellular senescence despite expression of p16(Ink4a), p19(Arf), p53, and p21(Waf1/Cip1).

Primary cultured mouse hepatic cells become senescent within a short period, although rare cells form colonies from which continuously proliferating cell lines can be established. In contrast, hepatic tumor (HT) cells show little senescence and higher colony-forming capacity. To assess this difference, we investigated p16(Ink4a)/p19(Arf)/p53/p21(Waf1/Cip1) expression in primary normal and HT cells, together with cell lines established from both. In primary normal cells, p16(Ink4a)/p19(Arf) were expressed only in association with senescence and disappeared at later stages of colony formation. In contrast, primary HT cells showed sustained p16(Ink4a)/p19(Arf) expression from the beginning. No p16(Ink4a)/p19(Arf) alterations, such as deletion, mutations, or hypermethylation, were detected in the primary HT cells, although most cell lines derived from either normal or HT cell colonies lost p16(Ink4a) or p19(Arf) expression owing to hypermethylation or homozygous deletion of p16(Ink4a)/p19(Arf). On the other hand, primary normal and HT cells and most cell lines showed constitutively elevated expression of p53/p21(Waf1/Cip1), with a further increment after ultraviolet ir-radiation, indicating a functionally normal p53 pathway. These results indicate that primary HT cells are resistant to senescence despite retaining p16(Ink4a)/p19(Arf)/p53/p21(Waf1/Cip1) expression and that loss of p16(Ink4a)/p19(Arf) function is associated only with establishment of the cell lines.

Alkylating Agents↗

PubMed2001

Cytotoxic effect of an anti-liver monoclonal autoantibody obtained after neonatal thymectomy in mice.

A monoclonal autoantibody, LSA-1, against murine liver antigen was obtained by fusing spleen cells from a neonatally thymectomized BALB/c mouse with SP2/0 murine myeloma cells. The LSA-1 isotype was IgG2b and kappa. LSA-1 was specific to the liver, especially, to a liver-specific membrane lipoprotein (LSP) fraction. By Western blotting analysis, LSA-1 mainly detected a 100 kDa protein of LSP fraction. LSA-1 stained cytoplasm of the cryostat sections of liver in immunohistochemical analysis. Furthermore, the antigen recognized with LSA-1 was highly expressed on the surface of a murine hepatoma cell line, MH134, slightly on a murine normal liver cell line, C1469, and on freshly prepared hepatocytes, but not on spleen cells. LSA-1 had a cytotoxic activity on liver cell lines in the presence of a complement in vitro. Furthermore, injection of LSA-1 into mice-induced liver injury. These results suggest that anti-liver autoantibody plays an important role in the induction of autoimmune hepatitis. Accordingly, this antibody will be a useful tool for the analysis of the pathogenesis of autoimmune hepatitis.

Animals↗

PubMed2001

Treatment of myelodysplastic syndrome with low-dose human granulocyte colony-stimulating factor: a multicenter study.

The objective of this study was to determine the hematopoietic effects and toxicity of low-dose granulocyte colony-stimulating factor (G-CSF) in myelodysplastic syndrome (MDS) patients with neutropenia. Recombinant human G-CSF (Lenograstim) was administered by daily subcutaneous injection with an initial dosage of 0.5 microg/kg per day for 2 weeks. Patients not responding to the initial dosage received the escalated dosage, 1 to 2 microg/kg per day for 2 weeks. Eligibility criteria were the following: French-American-British disease classification subtype refractory anemia (RA), refractory anemia with ringed sideroblasts (RARS), or refractory anemia with excess blasts (RAEB) with an absolute neutrophil count (ANC) of <1.5 x 10(9)/L. Criteria indicating response to treatment were ANC of >1.5 x 10(9)/L and doubling of ANC on at least 2 occasions. Thirty-two MDS patients were recruited from 6 university hospitals. Eighteen patients had RA, 4 had RARS, and 10 had RAEB. Median age was 56.4 years (range, 28-87 years). Twenty-six patients (81.2%) had an increase in ANC from a median of 0.94+/-0.35 x 10(9)/L to 4.24+/-3.78 x 10(9)/L. Three of 6 patients who did not respond to the initial dosage responded to the escalated dosage of 1 microg/kg per day. Eighteen (81.8%) of 22 patients with RA or RARS responded compared with 8 (80%) of 10 patients with RAEB. The response rates in patients with ANCs of <0.5 x 10(9)/L. 0.5 to <1.0 x 10(9)/L, and 1.0 to 1.5 x 10(9)/L were 80%, 70%, and 88.2%, respectively. The side effects were minimal. No significant changes in hemoglobin levels or platelet counts were observed. In conclusion, low-dose G-CSF administered by subcutaneous injection is well tolerated and effective in improving neutropenia in MDS patients.

Adult↗