Biomedical subjects

Minoru Yoshida

Publications and source records attributed to Minoru Yoshida.

At least 37 records · Page 2Linked to original sources

PubMed2006

Nucleocytoplasmic transport of fluorescent mRNA in living mammalian cells: nuclear mRNA export is coupled to ongoing gene transcription.

In eukaryotic cells, export of mRNA from the nucleus to the cytoplasm is one of the essential steps in gene expression. To examine mechanisms involved in the nucleocytoplasmic transport of mRNA, we microinjected fluorescently labeled fushi tarazu (ftz) pre-mRNA into the nuclei of HeLa cells. The injected intron-containing ftz pre-mRNA was distributed to the SC35 speckles and exported to the cytoplasm after splicing by an energy-requiring active process. In contrast, the injected intron-less ftz mRNA was diffusely distributed in the nucleus and then presumably degraded. Interestingly, export of the ftz pre-mRNA was inhibited by treatment with transcriptional inhibitors (actinomycin D, alpha-amanitin or DRB). Cells treated with transcriptional inhibitor showed foci enriched with the injected mRNA, which localize side by side with SC35 speckles. Those nuclear foci, referred to as TIDRs (transcriptional-inactivation dependent RNA domain), do not overlap with paraspeckles. In addition, in situ hybridization analysis revealed that the export of endogenous poly(A)+ mRNA is also affected by transcriptional inactivation. These results suggest that nuclear mRNA export is coupled to ongoing gene transcription in mammalian cells.

Active Transport, Cell Nucleus↗

PubMed2006

Induction of a ribotoxic stress response that stimulates stress-activated protein kinases by 13-deoxytedanolide, an antitumor marine macrolide.

13-Deoxytedanolide is a structurally unique macrolide with strong antitumor activity isolated from a marine sponge. Recently, we showed that 13-deoxytedanolide bound to the large subunit of the yeast ribosome and inhibited polypeptide elongation in vitro, but the mechanism by which it exerts antitumor activity is still unknown. Here we show that 13-deoxytedanolide strongly induces plasminogen activator inhibitor 1 (PAI-1) promoter-derived gene expression. 13-Deoxytedanolide, unlike TGF-beta, did not cause apparent nuclear translocation of Smad2/3, but it relocalized the temperature-sensitive mutant of mouse p53 (p53Val153) from the cytoplasm to the nucleus at a nonpermissive temperature, suggesting that 13-deoxytedanolide inhibits protein synthesis. Indeed, the drug inhibited in vivo protein synthesis at low nanomolar concentrations and strongly activated stress-activated protein kinases such as p38 mitogen-activated protein kinase and Jun NH2-terminal protein kinase (JNK). Anisomycin, a well-known inducer of ribotoxic stress that activates both p38 and JNK, also activated PAI-1 gene expression, while other protein synthesis inhibitors that do not activate the kinases failed to do so. PAI-1 gene expression by 13-deoxytedanolide and anisomycin was blocked by SB202190, a specific inhibitor of p38, and SP600125, an inhibitor of both p38 and JNK. 13-Deoxytedanolide and anisomycin caused activation of apoptosis signal-regulating kinase 1, MKK3/MKK6, and SEK1/MKK4, the regulatory kinases upstream of p38 and JNK. These results suggest that 13-deoxytedanolide, like anisomycin, triggers a ribotoxic stress response that activates stress-activated protein kinase cascades, thereby inducing PAI-1 gene expression and apoptosis.

Animals↗

PubMed2006

[Bilateral adrenal masses from renal cell carcinoma: a case report].

We report a case of bilateral adrenal metastasis from renal cell carcinoma. A 65-year-old man was referred to our hospital for a right renal mass. A computed tomography revealed a 9 cm right renal tumor and bilateral adrenal masses (3.5 cm on the right side and 4.5 cm on the left). A right radical nephrectomy and bilateral adrenalectomy demonstrated renal cell carcinoma with metastasis to bilateral adrenal glands. The pathological findings of the right renal tumor showed clear cell carcinoma, G3 > G2 and both adrenal tumors showed the same pathology as the right renal tumor. The patient is alive with lung metastasis after 15 months postoperatively treated with interferon-alpha.

Adrenal Gland Neoplasms↗

PubMed2006

Rapid progression of carcinoma of the penis after laser treatment.

We report a case of rapid progression of carcinoma of the penis, in a 90-year-old man, one year after yttrium-aluminum-garnet (YAG) laser therapy. This patient had received YAG laser ablation for well-differentiated, verrucous, squamous cell carcinoma of the penis at the age of 89, and was found on physical examination to have local recurrence. The recurrent tumors were surgically resected, and the histological examination revealed infiltrating, poorly-differentiated squamous cell carcinoma. The patient developed metastases of bilateral inguinal lymph nodes and liver and died of penile carcinoma. We should be aware of the possibility of an unfavorable outcome following the use of laser therapy against carcinoma of the penis.

Aged, 80 and over↗

PubMed2006

Leptomycin B, a metabolite of Streptomyces, inhibits the expression of inducible nitric oxide synthase in BV2 microglial cells.

Overexpression of inducible nitric oxide synthase (iNOS) and the resultant overproduction of NO has been implicated in neuronal inflammatory diseases. Leptomycin B (LMB), a metabolite of Streptomyces, has been identified as a specific inhibitor of CRM1 nuclear export receptor. In this study, we evaluated the effect of LMB on lipopolysaccharide (LPS)-induced iNOS expression in BV2 cells, a murine microglial cells and the associated mechanisms. LMB strongly inhibited LPS-induced iNOS protein and mRNA expressions in BV2 cells in which 10 ng/ml of LMB (18 nM) was sufficient to greatly down-regulate iNOS by LPS, suggesting the potency of LMB to inhibit iNOS. The data of iNOS promoter-driven luciferase assay further suggested that the LMB inhibitory effect was in part due to inhibition of iNOS transcription. However, LPS-induced activation of various intracellular signaling proteins, such as nuclear factor-kappaB (NF-kappaB), extracellular signal-regulated kinases (ERKs), p38s, and c-Jun N-terminal kinases (JNKs), whose activations are known to be important for iNOS expression by LPS in BV2 cells, were not affected in the presence of LMB. Together, these results suggest that LMB inhibits iNOS expression in response to LPS in BV2 microglia, and the inhibition seems to be associated with blockage of CRM1-mediated iNOS mRNA nuclear export and also in part transcriptional down-regulation of iNOS, but not through modulation of NF-kappaB and the mitogen-activated protein kinase signaling pathways.

Active Transport, Cell Nucleus↗

PubMed2005

Design and synthesis of phthalimide-type histone deacetylase inhibitors.

Several hydroxamic acid derivatives with a substituted phthalimide group as a linker and/or cap structure, prepared during structural development studies based on thalidomide, were found to have histone deacetylase (HDAC)-inhibitory activity. Structure-activity relationship studies indicated that nature of the substituent introduced at the phthalimide nitrogen atom, introduction of a hydroxamic acid structure, and distance between the N-hydroxyl group and the cap structure are important for HDAC-inhibitory activity.

Enzyme Inhibitors↗

PubMed2005

Behavioral changes in metallothionein-null mice after the cessation of long-term, low-level exposure to mercury vapor.

The neurobehavioral changes in wild-type and metallothionein (MT)-null mice after the cessation of long-term, low-level exposure to Hg0 were investigated. MT-null and wild-type females were continuously (24 h/day) exposed to mercury vapor (Hg0) at 0.055 mg/m3 (range: 0.043-0.073 mg/m3), which was similar to the current threshold limit value (TLV), for 29 weeks. The effects on behavior, such as locomotor activity in the open field (OPF), learning ability in the passive avoidance response (PA) and spatial learning ability in the Morris water maze (MM) were examined immediately and 12 weeks after the cessation of exposure. Immediately after the exposure had ceased, total locomotor activity in OPF was decreased in the both strain of mice, although the MT-null mice appeared to show more distinct effect. In the PA test, the exposed animals of both strains showed learning impairment as compared to un-exposed mice. Twelve weeks after the cessation of exposure, the locomotor activity in OPF was elevated in the exposed mice of both strains, while the learning ability in the PA test appeared normal in both strains. Spatial learning ability was not affected at all. Immediately after the exposure had ceased, the brain mercury concentration of the exposed wild-type mice was 1.75 microg/g, twofold of that in the MT-null mice. In 12 weeks, brain mercury levels decreased to approximately 1/20 of those in immediately after the exposure in both of the strains. These results for the first time indicated that long-term, low-level exposure to Hg0 could exert neurobehavioral effects, which were not reversible even after a long exposure-free period. Whereas the effects on learning ability were presumably transient, the effects on spontaneous behavior as evaluated in OPF were persistent. Finally, the MT-null mice seemed more susceptible to Hg0-induced neurotoxicity than the wild-type mice, confirming our previous results.

Animals↗

PubMed2005

Measurement of radical-scavenging ability in hepatic metallothionein of rat using in vivo electron spin resonance spectroscopy.

In this study, the ability of metallothionein (MT) to scavenge free radicals was determined by in vivo electron spin resonance (ESR) spectroscopy using a carbamoyl-PROXYL, nitroxyl radical, as a spin probe. Production of metallothionein was induced in the liver of rats with ZnSO(4) (0.2 mol/kg, ip) and the intensity of the carbamoyl-PROXYL ESR signal was measured at the upper abdominal level which is a position of the liver. After the injection of carbamoyl-PROXYL, the peak of ESR signal gradually decreased and showed a linear decay curve. The rate of decay of carbamoyl-PROXYL, the spin clearance rate, was determined over the first 3 min. The spin clearance rate did not differ significantly between ZnSO(4)-treated and control rats. When rats were fasted for 24 h, hepatic glutathione (GSH) concentrations decreased significantly and the spin clearance rate was significantly lower than non-fasted rats. However, the spin clearance rate of the fasted rats treated with Zn returned to the control level. To reduce GSH concentrations in the liver, buthionine sulfoximine (BSO, 2 mmol/kg, ip) was injected into the rats. The spin clearance rate of rats treated with BSO was significantly decreased as compared with that of control rats without BSO treatment. In rats treated with Zn, the decay rate of carbamoyl-PROXYL increased significantly in spite of the depletion of the hepatic GSH caused by BSO treatment, and returned to the control level. These results indicate that when the hepatic GSH concentration was significantly decreased by fasting and the administration of BSO, hepatic MT acted as a scavenger of free radicals. We suggest that GSH and MT act cooperatively as antioxidants to scavenge free radicals produced in response to various forms of stress, and MT serves as a second rather than the first line of defense.

Animals↗

PubMed2005

HIV-1 Tat targets Tip60 to impair the apoptotic cell response to genotoxic stresses.

HIV-1 transactivator Tat uses cellular acetylation signalling by targeting several cellular histone acetyltransferases (HAT) to optimize its various functions. Although Tip60 was the first HAT identified to interact with Tat, the biological significance of this interaction has remained obscure. We had previously shown that Tat represses Tip60 HAT activity. Here, a new mechanism of Tip60 neutralization by Tat is described, where Tip60 is identified as a substrate for the newly reported p300/CBP-associated E4-type ubiquitin-ligase activity, and Tat uses this mechanism to induce the polyubiquitination and degradation of Tip60. Tip60 targeting by Tat results in a dramatic impairment of the Tip60-dependent apoptotic cell response to DNA damage. These data reveal yet unknown strategies developed by HIV-1 to increase cell resistance to genotoxic stresses and show a role of Tat as a modulator of cellular protein ubiquitination.

Acetyltransferases↗

PubMed2005

Significance of HDAC6 regulation via estrogen signaling for cell motility and prognosis in estrogen receptor-positive breast cancer.

Histone deacetylase (HDAC) 6 is a subtype of the HDAC family; it deacetylates alpha-tubulin and increases cell motility. Here, we investigate the impact of an alteration of HDAC6 expression in estrogen receptor alpha (ER)-positive breast cancer MCF-7 cells, as we identified that HDAC6 is a novel estrogen-regulated gene. MCF-7 treated with estradiol showed increased expression of HDAC6 mRNA and protein and a four-fold increase in cell motility in a migration assay. Cell motility was increased to the same degree by stably transfecting the HDAC6 expression vector into MCF-7 cells. In both cases, the cells changed in appearance from their original round shape to an axon-extended shape, like a neuronal cell. This HDAC6 accumulation caused the deacetylation of alpha-tubulin. Either the selective estrogen receptor modulator tamoxifen (TAM) or the pure antiestrogen ICI 182,780 prevented estradiol-induced HDAC6 accumulation and deacetylation of alpha-tubulin, leading to reduced cell motility. Tubacin, an inhibitory molecule that binds to the tubulin deacetylation domain of HDAC6, also prevented estradiol-stimulated cell migration. Finally, we evaluated HDAC6 protein expression in 139 consecutively archived human breast cancer tissues by immunohistochemical staining. The prognostic analyses for these patients revealed no significant differences based on HDAC6 expression. However, subset analysis of ER-positive patients who received adjuvant treatment with TAM (n = 67) showed a statistically significant difference in relapse-free survival and overall survival in favor of the HDAC6-positive group (P < 0.02 and P < 0.05, respectively). HDAC6 expression was an independent prognostic indicator by multivariate analysis (odds ratio = 2.82, P = 0.047). These results indicate the biological significance of HDAC6 regulation via estrogen signaling.

Anilides↗

PubMed2005

HDAC6 regulates Hsp90 acetylation and chaperone-dependent activation of glucocorticoid receptor.

The molecular chaperone heat shock protein 90 (Hsp90) and its accessory cochaperones function by facilitating the structural maturation and complex assembly of client proteins, including steroid hormone receptors and selected kinases. By promoting the activity and stability of these signaling proteins, Hsp90 has emerged as a critical modulator in cell signaling. Here, we present evidence that Hsp90 chaperone activity is regulated by reversible acetylation and controlled by the deacetylase HDAC6. We show that HDAC6 functions as an Hsp90 deacetylase. Inactivation of HDAC6 leads to Hsp90 hyperacetylation, its dissociation from an essential cochaperone, p23, and a loss of chaperone activity. In HDAC6-deficient cells, Hsp90-dependent maturation of the glucocorticoid receptor (GR) is compromised, resulting in GR defective in ligand binding, nuclear translocation, and transcriptional activation. Our results identify Hsp90 as a target of HDAC6 and suggest reversible acetylation as a unique mechanism that regulates Hsp90 chaperone complex activity.

Acetylation↗

PubMed2005

Nuclear-cytoplasmic shuttling of a RING-IBR protein RBCK1 and its functional interaction with nuclear body proteins.

The intracellular localization of a RING-IBR protein, RBCK1, possessing DNA binding and transcriptional activities, has been investigated. The endogenous RBCK1 was found in both the cytoplasm and nucleus. Particularly in the nucleus, it was localized in the granular structures, most likely nuclear bodies. In contrast, the over-expressed RBCK1 was detected exclusively in the cytoplasm. When the cells were treated with leptomycin B, the over-expressed RBCK1 accumulated in the nuclear bodies. These results suggest that RBCK1 possesses the signal sequences responsible for the nuclearcytoplasmic translocation. Mutational analysis of RBCK1 has indicated that an N-terminal region containing Leu-142 and Leu-145 and a C-terminal one containing the RING-IBR domain serve as the nuclear export and localization signals, respectively. Thus, RBCK1 is a transcription factor dynamically shuttling between cytoplasm and nucleus. Furthermore, RBCK1 was found to interact with nuclear body proteins, CREB-binding protein (CBP), and promyelocytic leukemia protein (PML). Coexpression of RBCK1 with CBP significantly enhanced the transcriptional activity of RBCK1. Although PML per se showed no effect on the transcriptional activity of RBCK1, the CBP-enhanced activity was repressed by coexpression with PML, presumably through the interaction of PML and CBP. Taken together, our data demonstrate that RBCK1 is involved in transcriptional machinery in the nuclear bodies, and its transcriptional activity is regulated by nucleocytoplasmic shuttling.

Active Transport, Cell Nucleus↗

PubMed2005

Tiludronate inhibits prostaglandin F2alpha-induced vascular endothelial growth factor synthesis in osteoblasts.

We have previously reported that prostaglandin F2alpha (PGF2alpha) activates p44/p42 mitogen-activated protein (MAP) kinase through protein kinase C (PKC), resulting in the synthesis of vascular endothelial growth factor (VEGF) in osteoblast-like MC3T3-E1 cells, and that incadronate, a bisphosphonate, amplifies the VEGF synthesis. In the present study, we investigated the effects of tiludronate and etidronate, other bisphosphonates, on the PGF2alpha-stimulated VEGF synthesis in these cells. Tiludronate reduced the synthesis of VEGF induced by PGF2alpha. The PGF(2alpha)-stimulated phosphorylation of p44/p42 MAP kinase was suppressed by tiludronate. On the other hand, etidronate affected neither the VEGF synthesis nor the phosphorylation of p44/p42 MAP kinase elicited by PGF2alpha. Tiludronate attenuated the phosphorylation of both Raf-1 and MEK1/2 induced by PGF2alpha. The VEGF synthesis stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA), a direct activator of PKC, was suppressed by tiludronate. The TPA-induced phosphorylations of Raf-1, MEK1/2 and p44/p42 MAP kinase were inhibited by tiludronate. These results strongly suggest that tiludronate but not etidronate suppresses the PGF2alpha-stimulated VEGF synthesis in osteoblasts, and that the effect of tiludronate is exerted at the point between PKC and Raf-1.

3T3 Cells↗

PubMed2005

Neurobehavioral changes in metallothionein-null mice prenatally exposed to mercury vapor.

We studied the neurobehavioral effects of prenatal exposure of MT-null and wild-type mice to elemental mercury vapor (Hg0). Pregnant mice of both strains were repeatedly exposed to Hg0 vapor at 0.50 and 0.56 mg/m3 for 6 h/day until the 18th day of gestation. The behavioral effects were evaluated with locomotor activity in the open field, learning ability in the passive avoidance response and spatial learning ability in the Morris water maze at 12 weeks of age. Hg0-exposed MT-null mice showed a significant decrease in total locomotor activity in males, and a learning disability in the passive avoidance response and a retarded acquisition in the Morris water maze in females as compared with the control. In contrast, Hg0-exposed wild-type mice did not differ from controls in the three behavioral measurements. The results indicate that MT-null mice would be more susceptible than wild-type mice to the behavioral neurotoxicity of prenatal Hg0 exposure. Mercury concentrations in the brain of both strains were slightly higher in the exposed group than in the control group, indicating the retention of residual mercury even 12 weeks after the cessation of the exposure. Brain concentrations of mercury were also significantly higher in the exposed-females than exposed-males in either strain. From these results, we suggest that the increased susceptibility of MT-null females to behavioral changes caused by prenatal Hg0 exposure is due to a greater retention of mercury and lack of MT-I,-II in the brain.

Animals↗

PubMed2005

13-Deoxytedanolide, a marine sponge-derived antitumor macrolide, binds to the 60S large ribosomal subunit.

13-Deoxytedanolide is a potent antitumor macrolide isolated from the marine sponge Mycale adhaerens. In spite of its remarkable activity, the mode of action of 13-deoxytedanolide has not been elucidated. [11-3H]-(11S)-13-Deoxydihydrotedanolide derived from the macrolide was used for identifying the target molecule from the yeast cell lysate. Fractionation of the binding protein revealed that the labeled 13-deoxytedanolide derivative strongly bound to the 80S ribosome as well as to the 60S large subunit, but not to the 40S small subunit. In agreement with this observation, 13-deoxytedanolide efficiently inhibited the polypeptide elongation. Interestingly, competition studies demonstrated that 13-deoxytedanolide shared the binding site on the 60S large subunit with pederin and its marine-derived analogues. These results indicate that 13-deoxytedanolide is a potent protein synthesis inhibitor and is the first macrolide to inhibit the eukaryotic ribosome.

Animals↗

PubMed2005

Adenylyl cyclase-cAMP system inhibits thyroid hormone-stimulated osteocalcin synthesis in osteoblasts.

It is generally recognized that thyroid hormone modulates osteoblast cell function. We have previously shown that triiodothyronine (T(3)) activates p38 mitogen-activated protein (MAP) kinase, resulting in the synthesis of osteocalcin in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of the adenylyl cyclase-cAMP system on thyroid hormone-stimulated osteocalcin synthesis in these cells. Dibutyryl-cAMP (DBcAMP) reduced the osteocalcin synthesis stimulated by T(3). Forskolin and cholera toxin suppressed the osteocalcin synthesis while dideoxyforskolin, a forskolin derivative that does not activate adenylyl cyclase, had little effect on the synthesis. KT5720, a selective inhibitor of protein kinase A, reversed the inhibitory effect of forskolin or DBcAMP. DBcAMP and forskolin markedly reduced the phosphorylation of p38 MAP stimulated by T(3). Pituitary adenylate cyclase-activating polypeptide (PACAP) significantly inhibited the T(3)-stimulated osteocalcin synthesis. These results strongly suggest that the adenylyl cyclase-cAMP system has an inhibitory role in thyroid hormone-stimulated osteocalcin synthesis via suppression of p38 MAP kinase activation in osteoblasts.

Adenylyl Cyclases↗

PubMed2005

Proteomic analysis of organ-specific post-translational lysine-acetylation and -methylation in mice by use of anti-acetyllysine and -methyllysine mouse monoclonal antibodies.

Post-translational lysine-acetylation and -methylation are two major PTMs of lysine residues in proteins. Recently, we established pan-reactive anti-acetyllysine mouse mAbs, which can bind to Nepsilon-acetylated lysine residues in various contexts of amino acid sequences. In the present study, we established pan-reactive anti-methyllysine mouse mAbs comparable to the anti-acetyllysine ones. By using these anti-acetyllysine and -methyllysine antibodies, we found that the pattern of lysine-acetylated and -methylated proteins in mouse organs showed extreme variation from organ to organ. We selected brain and skeletal muscle as model cases to be further analyzed by 2-DE followed by Western blotting. In brain, alpha-tubulin at its basal level was found to be extremely acetylated; and alpha-enolase was shown to be a newly recognized possibly acetylated protein. NF-L protein, Hsc70, alpha-tubulin fragments, beta-actin, and brain-type creatine kinase were identified as putative lysine-methylated proteins in mouse brain. In skeletal muscle, lysine-methylation of alpha-actin and both lysine-acetylation and -methylation of muscle-type creatine kinase were found as novel putative lysine-modified proteins. The approach presented here might be useful to find novel disease markers and/or drug target molecules that would not be noticed by use of the traditional proteomic approach only.

Acetylation↗

PubMed2005

Current status of diagnosis and treatment of invasive fungal infections in Japan: the influence of the new Japanese guidelines.

One year after the release of the Japanese "Guidelines for the Diagnosis and Treatment of Deep-Seated Mycosis" we conducted a survey of clinicians to determine the extent to which the new guidelines had penetrated clinical practice, and compared these results with those of a previous survey. The responses to the current survey regarding the diagnosis and treatment had changed from those of the previous survey to reflect the new recommendations, showing that the guidelines have had an effect on clinical practice. However, the current survey highlights a need to provide more practical information in the guidelines for use in the clinical settings. In particular, physicians expect the guidelines to proactively provide more information about the features of both current and new drugs. In addition, an effective drug against the genus Aspergillus is eagerly awaited. However, because it is difficult to differentiate among filamentous fungi, there is a need for a drug with broad-spectrum coverage against filamentous fungi. Investigation of combination therapy consequently becomes necessary. Definitive diagnoses of invasive fungal infection are too scarce at the national level. The cooperation of clinicians for organizing more definitive diagnoses would be appreciated when the guidelines are revised.

Antifungal Agents↗