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Biomedical subjects

M Yoshida

Publications and source records attributed to M Yoshida.

At least 163 records · Page 9Linked to original sources
PubMed2001

Biliary excretion of polystyrene microspheres depends on the type of receptor-mediated uptake in rat liver.

Hepatic uptake and biliary excretion of fluorescein isothiocyanate-labeled polystyrene microspheres with a particle size of 50 nm (MS-50) were studied in rats. Liver perfusion studies revealed that not only apo-E-mediated but also asialoglycoprotein receptor-mediated uptake is involved in the mechanism of the serum protein-dependent uptake of MS-50 in the liver. The uptake of MS-50 mediated by apo-E contributes more to the total uptake of MS-50 by the hepatocytes than that via asialoglycoprotein receptor in the presence of serum in the perfusate. Furthermore, it was found that MS-50 is substantially excreted into the bile by transcytosis. The extent of exocytosis of MS-50 taken up by the hepatocytes was much higher after MS-50 was endocytosed via asialoglycoprotein receptor than after taken up via the process mediated by apo-E. On the basis of these results, a possible regulation of the intracellular sorting of ligands, depending on the receptor-mediated uptake mechanism, was inferred.

Animals↗
PubMed2001

Nuclear export of human beta-catenin can occur independent of CRM1 and the adenomatous polyposis coli tumor suppressor.

beta-Catenin is a mediator of the Wnt-signaling pathway. In many cancers, beta-catenin is stabilized and accumulates in the nucleus where it associates with lymphoid-enhancing factor 1/ T-cell transcription factors to activate genes involved in cell transformation. Previously, we showed that adenomatous polyposis coli (APC) protein can regulate beta-catenin localization by nuclear export. In this study, we used in vitro transport assays to test whether cellular beta-catenin can exit the nucleus independent of APC and the CRM1 export receptor. In digitonin-permeabilized SW480 (APC(mut/mut)) tumor cells, nuclear beta-catenin decreased >60% in export reactions in the absence of exogenous factors. Under similar conditions, nuclear c-ABL was only exported after the addition of cytosolic extract, and the export was blocked by the CRM1-specific inhibitor, leptomycin B. The nuclear export of beta-catenin was not blocked by leptomycin B treatment, revealing a CRM1- and APC-independent pathway. The export of beta-catenin was sensitive to lower temperatures and the removal of ATP, indicating an active process. Ectopically expressed yellow fluorescent protein-beta-catenin also displayed CRM1-independent export. Conversely, the overexpression of the CRM1 transporter moderately stimulated export of nuclear beta-catenin, confirming that beta-catenin exits the nucleus by at least two distinct pathways. The shuttling ability of tumor cell beta-catenin has implications for its regulation and its role in transferring signals between the nucleus and plasma membrane.

Adenomatous Polyposis Coli Protein↗
PubMed2001

Time-dependent promotion activity of 17beta-estradiol on uterine carcinogenesis in mice initiated with N-ethyl-N-nitrosourea.

The time-dependent promotion activity of 17beta-estradiol (E2) by initiation with N-ethyl-N-nitrosourea (ENU) on induction of mouse uterine endometrial proliferative lesions was examined. Illumination-induced persistent estrous female CD-1 mice were divided into five groups at 9 weeks of age. At 10 weeks of age, mice in all groups (n=25) were given a single intra-uterine administration of ENU (50 mg/kg), dissolved in polyethylene glycol. Animals in Groups 2 to 5 were then implanted s.c. with an E2 pellet at 9, 11, 14 and 17 weeks of age. The implants were left in place for 8 weeks and then taken out. At the termination of the experiment (week 15 after the ENU-treatment), all surviving mice were killed and the development of uterine proliferative lesions were assessed. All groups demonstrated endometrial hyperplasias and adenocarcinomas and the incidences of the latter in ENU plus E2 treated animals (Groups 2 to 5; 36, 48, 35 and 36%, respectively) were significantly higher compared to 8% for Group 1, without any variation with the age at E2 treatment. However, the incidences of adenocarcinomas plus severe hyperplasias increased from Groups 1 to 5 (28, 40, 56; P<0.05, 61; P<0.05 and 80%; P<0.01, respectively), indicating that promotion effects of E2 on induction of uterine proliferative lesions in the uterine endometrium become more pronounced with the interval after ENU initiation.

Adenocarcinoma↗
PubMed2001

Resolution of distinct rotational substeps by submillisecond kinetic analysis of F1-ATPase.

The enzyme F1-ATPase has been shown to be a rotary motor in which the central gamma-subunit rotates inside the cylinder made of alpha3beta3 subunits. At low ATP concentrations, the motor rotates in discrete 120 degrees steps, consistent with sequential ATP hydrolysis on the three beta-subunits. The mechanism of stepping is unknown. Here we show by high-speed imaging that the 120 degrees step consists of roughly 90 degrees and 30 degrees substeps, each taking only a fraction of a millisecond. ATP binding drives the 90 degrees substep, and the 30 degrees substep is probably driven by release of a hydrolysis product. The two substeps are separated by two reactions of about 1 ms, which together occupy most of the ATP hydrolysis cycle. This scheme probably applies to rotation at full speed ( approximately 130 revolutions per second at saturating ATP) down to occasional stepping at nanomolar ATP concentrations, and supports the binding-change model for ATP synthesis by reverse rotation of F1-ATPase.

Adenosine Triphosphate↗
PubMed2001

Regulation of vascular endothelial growth factor transcription by endothelial PAS domain protein 1 (EPAS1) and possible involvement of EPAS1 in the angiogenesis of renal cell carcinoma.

BACKGROUND: Endothelial PAS domain protein 1 (EPAS1) is a basic helix-loop-helix/PAS domain transcription factor that expressed most abundantly in highly vascularized organs. The authors examined the effect of transfection of EPAS1 cDNA on the endogenous expression of vascular endothelial growth factor (VEGF) in the 293 Tet-Off cell line and the possible involvement of EPAS1 in the angiogenesis of renal cell carcinoma (RCC). METHODS: Complete cDNA of EPAS1 was cloned and transfected to cells from the 293 Tet-Off fetal kidney cell line, in which the expression of EPAS1 can be inhibited by doxycycline. The subsequent changes in expression pattern of VEGF and transferrin receptor (TfR), a target gene of hypoxia-inducible factor 1alpha (HIF-1alpha), were examined by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay. In addition, expression of EPAS1, HIF-1alpha, and VEGF were analyzed by semiquantitative RT-PCR in five RCC cell lines and in 13 RCC tissue samples. In situ hybridization was performed on 7 of the 13 RCC tissue samples. RESULTS: Endogenous VEGF was increased significantly by the introduction of EPAS1 cDNA at both the mRNA level and the protein level. With the inhibition of EPAS1 by doxycycline treatment, the expression of VEGF was significantly decreased accordingly, whereas the expression of TfR was not affected. EPAS1 was detected in all of the RCC cell lines examined. In RCC tissue samples, EPAS1 mRNA and VEGF mRNA were increased significantly in tumor tissues compared with normal adjacent kidney tissues. In situ hybridization showed that EPAS1 and VEGF were coexpressed topographically in tumor tissues. CONCLUSIONS: These results suggest that endogenous VEGF can be up-regulated transcriptionally by EPAS1, and EPAS1 may be involved in the angiogenesis of RCC.

Anti-Bacterial Agents↗
PubMed2001

Effects of neonatal exposure to a high-dose p-tert-octylphenol on the male reproductive tract in rats.

Time-course alterations in morphological changes of the reproductive tract including spermatogenesis as well as pituitary and gonadal hormones, reproductive ability, and the size of the sexually dimorphic nucleus of the preoptic area (SDN-POA) were investigated in male rats neonatally exposed to 100 mg/kg p-tert-octylphenol (OP) subcutaneously. OP treatment affected hormone levels of follicle stimulating hormone (FSH) and testosterone, reproductive organ weights and sperm counts. Slightly depressed FSH levels at prepuberty and prolonged suppression of testosterone till 7 weeks of age were observed as two hormonal alterations. The lasting reduction in testosterone appeared to be associated with growth inhibition of male reproductive organs such as the testis, prostate and epididymis, these demonstrating low organ weights compared with those of age-matched controls till 7 weeks of age. The FSH concentrations after puberty showed a rise to values equal to or higher than those of the control group, suggesting recovery of maturation of the reproductive tract. No morphological abnormalities, even with morphometric stage analysis of spermatogenesis, were detected in the male reproductive tract throughout the study. Size of the SDN-POA and reproductive ability was comparable to those in controls. At the termination (18 weeks of age), however, a reduction in the sperm count in the epididymis of OP-treated animals demonstrated a possibility that the male reproductive system might be still affected by neonatal exposure to OP. The results observed demonstrate that neonatal exposure to a high-dose OP exerts estrogenic action directly or indirectly, resulting in slight but prolonged impairment of the male reproductive tract. The suppression of FSH caused by modulation of the hypothalamus-pituitary control system may be the trigger for the impairment, while the possibility of direct estrogenic action of OP is not ruled out. Our results also indicate that more sensitive endpoints should be established to detect the effects of neonatal exposure to estrogens or estrogenic compounds on the male reproductive tract.

Animals↗
PubMed2001

Hand-assisted laparoscopic total colorectal resection for familial adenomatous polyposis with coexisting rectal cancer.

When familial adenomatous polyposis (FAP) is diagnosed in a patient, prophylactic surgery must be performed whether colorectal cancer is present or not. Operations for FAP have been performed through a large median abdominal incision or an additional perineal incision, depending on the coexistence of rectal cancer. Recently, we reported a technique of laparoscopic rectal amputation without abdominal skin incision for patients with rectal cancer to minimize postoperative cardiac and respiratory complications [6]. In this article, we report a case of laparoscopically assisted proctocolectomy with ileostomy through a minimal abdominal and perineal skin incision performed by a hand-assisted procedure. The purpose of combining the perineal and laparoscopic approaches is to minimize the skin incision, while retaining a rate of cure and safety equivalent to those of conventional rectal amputation, by using the advantages of laparoscopic procedures, and to facilitate postoperative recovery and improve the quality of life for relatively young patients with FAP.

Adenomatous Polyposis Coli↗
PubMed2001

Interaction of the hepatitis B virus X protein with the Crm1-dependent nuclear export pathway.

The leucine-rich nuclear export signal (NES) is used to shuttle large cellular proteins from the nucleus to the cytoplasm. The nuclear export receptor Crm1 is essential in this process by recognizing the NES motif. Here, we show that the oncogenic hepatitis B virus (HBV) X protein (HBx) contains a functional NES motif. We found that the predominant cytoplasmic localization of HBx is sensitive to the drug leptomycin B (LMB), which specifically inactivates Crm1. Mutations at the two conserved leucine residues to alanine at the NES motif (L98A,L100A) resulted in a nuclear redistribution of HBx. A recombinant HBx protein binds to Crm1 in vitro. In addition, ectopic expression of HBx sequesters Crm1 in the cytoplasm. Furthermore, HBx activates NFkappaB by inducing its nuclear translocation in a NES-dependent manner. Abnormal cytoplasmic sequestration of Crm1, accompanied by a nuclear localization of NFkappaB, was also observed in hepatocytes from HBV-positive liver samples with chronic active hepatitis. We suggest that Crm1 may play a role in HBx-mediated liver carcinogenesis.

Amino Acid Sequence↗
PubMed2001

The role of the betaDELSEED motif of F1-ATPase: propagation of the inhibitory effect of the epsilon subunit.

In F(1)-ATPase, a rotary motor enzyme, the region of the conserved DELSEED motif in the beta subunit moves and contacts the rotor gamma subunit when the nucleotide fills the catalytic site, and the acidic nature of the motif was previously assumed to play a critical role in rotation. Our previous work, however, disproved the assumption (Hara, K. Y., Noji, H., Bald, D., Yasuda, R., Kinosita, K., Jr., and Yoshida, M. (2000) J. Biol. Chem. 275, 14260-14263), and the role of this motif remained unknown. Here, we found that the epsilon subunit, an intrinsic inhibitor, was unable to inhibit the ATPase activity of a mutant thermophilic F(1)-ATPase in which all of the five acidic residues in the DELSEED motif were replaced with alanines, although the epsilon subunit in the mutant F(1)-ATPase assumed the inhibitory form. In addition, the replacement of basic residues in the C-terminal region of the epsilon subunit by alanines caused a decrease of the inhibitory effect. Partial replacement of the acidic residues in the DELSEED motif of the beta subunit or of the basic residues in the C-terminal alpha-helix of the epsilon subunit induced a partial effect. We here conclude that the epsilon subunit exerts its inhibitory effect through the electrostatic interaction with the DELSEED motif of the beta subunit.

Adenosine Triphosphate↗
PubMed2001

Purine but not pyrimidine nucleotides support rotation of F(1)-ATPase.

The binding change model for the F(1)-ATPase predicts that its rotation is intimately correlated with the changes in the affinities of the three catalytic sites for nucleotides. If so, subtle differences in the nucleotide structure may have pronounced effects on rotation. Here we show by single-molecule imaging that purine nucleotides ATP, GTP, and ITP support rotation but pyrimidine nucleotides UTP and CTP do not, suggesting that the extra ring in purine is indispensable for proper operation of this molecular motor. Although the three purine nucleotides were bound to the enzyme at different rates, all showed similar rotational characteristics: counterclockwise rotation, 120 degrees steps each driven by hydrolysis of one nucleotide molecule, occasional back steps, rotary torque of approximately 40 piconewtons (pN).nm, and mechanical work done in a step of approximately 80 pN.nm. These latter characteristics are likely to be determined by the rotational mechanism built in the protein structure, which purine nucleotides can energize. With ATP and GTP, rotation was observed even when the free energy of hydrolysis was -80 pN.nm/molecule, indicating approximately 100% efficiency. Reconstituted F(o)F(1)-ATPase actively translocated protons by hydrolyzing ATP, GTP, and ITP, but CTP and UTP were not even hydrolyzed. Isolated F(1) very slowly hydrolyzed UTP (but not CTP), suggesting possible uncoupling from rotation.

Adenosine Triphosphate↗
PubMed2001

A comprehensive two-hybrid analysis to explore the yeast protein interactome.

Protein-protein interactions play crucial roles in the execution of various biological functions. Accordingly, their comprehensive description would contribute considerably to the functional interpretation of fully sequenced genomes, which are flooded with novel genes of unpredictable functions. We previously developed a system to examine two-hybrid interactions in all possible combinations between the approximately 6,000 proteins of the budding yeast Saccharomyces cerevisiae. Here we have completed the comprehensive analysis using this system to identify 4,549 two-hybrid interactions among 3,278 proteins. Unexpectedly, these data do not largely overlap with those obtained by the other project [Uetz, P., et al. (2000) Nature (London) 403, 623-627] and hence have substantially expanded our knowledge on the protein interaction space or interactome of the yeast. Cumulative connection of these binary interactions generates a single huge network linking the vast majority of the proteins. Bioinformatics-aided selection of biologically relevant interactions highlights various intriguing subnetworks. They include, for instance, the one that had successfully foreseen the involvement of a novel protein in spindle pole body function as well as the one that may uncover a hitherto unidentified multiprotein complex potentially participating in the process of vesicular transport. Our data would thus significantly expand and improve the protein interaction map for the exploration of genome functions that eventually leads to thorough understanding of the cell as a molecular system.

Fungal Proteins↗
PubMed2001

Identification of two rodent genes encoding homologues to seminal vesicle autoantigen: a gene family including the gene for prolactin-inducible protein.

We cloned two new paralogous genes that encode proteins homologous to seminal vesicle autoantigen (SVA) in rodents. The open reading frame of one mouse gene encodes a polypeptide consisting of 151 amino acid residues which has 43% identity to SVA. RT-PCR analysis showed selective expression in the colon, and thus the protein was tentatively named "SVA-like protein in the colon (SLP-C)". The other mouse gene has an open reading frame encoding 144 amino acid residues with 46 and 65% identity to SVA and SLP-C, respectively. Expression of this gene was detected in the mammary, submaxillary, parotid, and lacrimal glands, and this protein was named "SLP in the mammary gland (SLP-M)". Orthologs of both genes were also found in rats. The three homologous genes coding for SVA, SLP-C, and SLP-M may have been generated by gene duplication with divergence of tissue expression in the course of evolution. They comprise a unique structurally-related gene family. Moreover, these genes share significant sequence homology with that of another secretory glycoprotein, prolactin-inducible protein.

Amino Acid Sequence↗
PubMed2001

Early complications of high-dose methylprednisolone sodium succinate treatment in the follow-up of acute cervical spinal cord injury.

STUDY DESIGN: A prospective, randomized, and double-blind study comparing high-dose methylprednisolone sodium succinate (MPSS) with placebo, in the treatment of patients with acute cervical spinal cord injury. OBJECTIVES: To evaluate the complications of high-dose MPSS in patients with acute cervical spinal cord injury when administered within 8 hours of injury. SUMMARY OF BACKGROUND DATA: High-dose therapy with MPSS has been demonstrated to improve the recovery of motor function in patients with acute cervical spinal cord injury. However, little is known about the follow-up complications. METHODS: Forty-six patients, 42 men and 4 women (mean age, 60.6 years; range, 18-84), were included in the study: 23 in the MPSS group and 23 in the placebo group. They were treated without surgery for spinal cord injury in the cervical spine, and were enrolled in the trial if a diagnosis had been made and treatment had begun within 8 hours. Complications of high-dose therapy with MPSS were compared with placebo treatment throughout the study period and up to 2 months after injury. RESULTS: The MPSS group had 13 patients (56.5%) with complications, whereas the placebo group had 8 (34.8%). The difference between the two groups was not statistically significant (P = 0.139). There were eight instances of pulmonary complication with MPSS (34.8%) and one instance (4.34%) with placebo (P = 0.009). There were four instances of gastrointestinal complication (17.4%) with MPSS and none with placebo (P = 0.036). Pulmonary (complications were more prevalent in patients aged more than 60 years (P = 0.029). CONCLUSION: Aged patients with cervical spinal injury may be more likely to have pulmonary side effects (P = 0.029) after high-dose therapy with MPSS and thus deserve special care.

Adult↗
PubMed2001

Intermediate filament proteins define different glial subpopulations.

In certain species, specialized glial cells delineate cell domains in the central nervous system and assist in the elongation of axons by providing mechanical and chemical barriers. We showed previously, that the glial intermediate filament proteins vimentin and glial fibrillary acidic protein are extensively coexpressed in radial glia in the developing hindbrain, and that subsequently, the two proteins define distinct rhombomere domains: vimentin is localized in radial glia at the rhombomere boundaries and glial fibrillary acidic protein expression is restricted to the rhombomere centers (Yoshida and Colman [2000] J. Comp. Neurol. 424:47-57). The present study reveals that vimentin and glial fibrillary acidic protein continue to display distinct expression domains throughout the developing Xenopus central nervous system. Although the precise function of the two intermediate filaments in glial cells has yet to be revealed, the observations presented here suggests that glial intermediate filament proteins demarcate different populations of glial cells during nervous system development and that the existence of different glial populations may define glial boundaries.

Animals↗
PubMed2001

A role for Hsc70 in regulating nucleocytoplasmic transport of a temperature-sensitive p53 (p53Val-135).

Mouse temperature-sensitive p53(Val-135) accumulates in the nucleus and acts as a "wild-type" at 32 degrees C while it is sequestered in the cytoplasm at 37 degrees C. The cytoplasmic p53(Val-135) relocalized into the nucleus upon inhibition of the nuclear export at 37 degrees C, whereas a mutation in a major bipartite nuclear localization signal (NLS) caused constitutive cytoplasmic localization, indicating that it shuttled between the cytoplasm and the nucleus by its own nuclear export signal and NLS rather than tethered to cytoplasmic structures. Although the full-length p53(Val-135) did not bind the import receptor at 37 degrees C, a C-terminally truncated p53(Val-135) lacking residues 326-390 did bind it. Molecular chaperones such as Hsc70 were associated with p53(Val-135) at 37 degrees C but not at 32 degrees C. When the nuclear export was blocked by leptomycin B, only a fraction lacking Hsc70 was specifically accumulated in the nucleus. Immunodepletion of Hsc70 from the reticulocyte lysate caused p53(Val-135) to bind the import receptor. This binding was blocked by supplying the cell extract containing Hsc70 but not by the addition of recombinant Hsc70 alone. We suggest that the association with the Hsc70-containing complex prevents the NLS from the access of the import receptor through the C-terminal region of p53(Val-135) at 37 degrees C, whereas its dissociation at 32 degrees C allows rapid nuclear import.

Amino Acid Sequence↗
PubMed2001

Polyamine enhancement of the synthesis of adenylate cyclase at the translational level and the consequential stimulation of the synthesis of the RNA polymerase sigma 28 subunit.

The effects of polyamines on the synthesis of various final sigma subunits of RNA polymerase were studied using Western blot analysis. Synthesis of final sigma(28) was stimulated 4.0-fold and that of final sigma(38) was stimulated 2.3-fold by polyamines, whereas synthesis of other final sigma subunits was not influenced by polyamines. Stimulation of final sigma(28) synthesis was due to an increase in the level of cAMP, which occurred through polyamine stimulation of the synthesis of adenylate cyclase at the level of translation. Polyamines were found to increase the translation of adenylate cyclase mRNA by facilitating the UUG codon-dependent initiation. Analysis of RNA secondary structure suggests that exposure of the Shine-Dalgarno sequence of mRNA is a prerequisite for polyamine stimulation of the UUG codon-dependent initiation.

Adenylyl Cyclases↗