Biomedical subjects

K Nakayama

Publications and source records attributed to K Nakayama.

At least 55 records · Page 3Linked to original sources

PubMed2001

A mouse knock-in model exposes sequential proteolytic pathways that regulate p27Kip1 in G1 and S phase.

The protein p27Kip1 is an inhibitor of cell division. An increase in p27 causes proliferating cells to exit from the cell cycle, and a decrease in p27 is necessary for quiescent cells to resume division. Abnormally low amounts of p27 are associated with pathological states of excessive cell proliferation, especially cancers. In normal and tumour cells, p27 is regulated primarily at the level of translation and protein turnover. Phosphorylation of p27 on threonine 187 (T187) by cyclin-dependent kinase 2 (Cdk2) is thought to initiate the major pathway for p27 proteolysis. To critically test the importance of this pathway in vivo, we replaced the murine p27 gene with one that encoded alanine instead of threonine at position 187 (p27T187A). Here we show that cells expressing p27T187A were unable to downregulate p27 during the S and G2 phases of the cell cycle, but that this had a surprisingly modest effect on cell proliferation both in vitro and in vivo. Our efforts to explain this unexpected result led to the discovery of a second proteolytic pathway for controlling p27, one that is activated by mitogens and degrades p27 exclusively during G1.

Animals↗

PubMed2001

Unexpected transcriptional induction of monocyte chemoattractant protein 1 by proteasome inhibition: involvement of the c-Jun N-terminal kinase-activator protein 1 pathway.

Proteasome inhibitors, the well-known inhibitors of NF-kappaB, are recently considered therapeutic agents for inflammation. However, the anti-inflammatory properties of these agents have not been fully evaluated. In this report we describe a novel effect of proteasome inhibitors on the expression of monocyte chemoattractant protein 1 (MCP-1) in mesangial cells. We found that proteasome inhibitor MG132 dose-dependently induced expression of MCP-1 at the transcriptional level. The stimulatory effect was similarly observed with other proteasome inhibitors (proteasome inhibitor 1 and lactacystin) and in other cell types (NRK fibroblasts). The 5'-flanking region of the MCP-1 gene contains multiple AP-1 sites. To explore the mechanisms involved, we examined the effects of proteasome inhibition on the AP-1 pathway. Northern blot analysis showed that MG132 rapidly induced the expression of c-jun, but not c-fos. Immunoblot analysis showed that MG132 prevented degradation of c-Jun protein. Kinase assay revealed that c-Jun N-terminal kinase (JNK) was rapidly activated by MG132. Consistent with these results, a reporter assay showed that AP-1 activity was up-regulated after treatment with MG132. Curcumin, a pharmacological inhibitor of the JNK-AP-1 pathway, abrogated the induction of MCP-1 by MG132. Similarly, stable transfection with a dominant-negative mutant of c-Jun attenuated both MG132-induced activation of AP-1 and expression of MCP-1. The transcriptional activation by proteasome inhibitors was observed not only in MCP-1, but also in other AP-1-dependent genes, including stromelysin and mitogen-activated protein kinase phosphatase 1. These data revealed that proteasome inhibition triggered the expression of MCP-1 and other genes via the multistep induction of the JNK-c-Jun/AP-1 pathway.

Animals↗

PubMed2001

Developmental changes in 5-hydroxytryptamine-induced rhythmic activity in the spinal cord of rat fetuses in vitro.

The roles played by glycine- and glutamate-mediated synaptic transmission in the generation of 5-hydroxytryptamine (5-HT)-induced rhythmic activity were examined in isolated spinal cord preparations from fetal rats. Bath application of 5-HT (0.1-30 microM) evoked rhythmic activity in lumbar ventral roots at and after E14.5. Bath application of strychnine (5 microM), a glycine-receptor antagonist, reduced the frequency of the rhythmic activity to 37% of control at E14.5. Although, kynurenate (4 mM), a glutamate-receptor antagonist, had little effect at this stage, it completely abolished the 5-HT-induced rhythmic activity at and after E18.5, when strychnine had little effect on the frequency. These results indicate that, at and shortly after its onset, the rhythmic activity is driven mainly by glycinergic rather than glutamatergic excitatory synaptic inputs, but that the latter become dominant later on.

Animals↗

PubMed2001

Significantly higher activity of a cytoplasmic hammerhead ribozyme than a corresponding nuclear counterpart: engineered tRNAs with an extended 3' end can be exported efficiently and specifically to the cytoplasm in mammalian cells.

Hammerhead ribozymes were expressed under the control of similar tRNA promoters, localizing transcripts either in the cytoplasm or the nucleus. The tRNA(Val)-driven ribozyme (tRNA-Rz; tRNA with extra sequences at the 3' end) that has been used in our ribozyme studies was exported efficiently into the cytoplasm and ribozyme activity was detected only in the cytoplasmic fraction. Both ends of the transported tRNA-Rz were characterized comprehensively and the results confirmed that tRNA-Rz had unprocessed 5' and 3' ends. Furthermore, it was also demonstrated that the activity of the exported ribozyme was significantly higher than that of the ribozyme which remained in the nucleus. We suggest that it is possible to engineer tRNA-Rz, which can be exported to the cytoplasm based on an understanding of secondary structures, and then tRNA-driven ribozymes may be co-localized with their target mRNAs in the cytoplasm of mammalian cells.

Base Sequence↗

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

Membrane binding and substrate access merge in cytochrome P450 7A1, a key enzyme in degradation of cholesterol.

To study membrane topology and mechanism for substrate specificity, we truncated residues 2-24 in microsomal cytochrome P450 7A1 (P450 7A1) and introduced conservative and nonconservative substitutions at positions 214-227. Heterologous expression in Escherichia coli was followed by investigation of the subcellular distribution of the mutant P450s and determination of the kinetic and substrate binding parameters for cholesterol. The results indicate that a hydrophobic region, comprising residues 214-227, forms a secondary site of attachment to the membrane in P450 7A1 in addition to the NH(2)-terminal signal-anchor sequence. There are two groups of residues at this enzyme-membrane interface. The first are those whose mutation results in more cytosolic P450 (Val-214, His-225, and Met-226). The second group are those whose mutation leads to more membrane-bound P450 (Phe-215, Leu-218, Ile-224, and Phe-227). In addition, the V214A, V214L, V214T, F215A, F215L, F215Y, L218I, L218V, V219T, and M226A mutants showed a 5-12-fold increased K(m) for cholesterol. The k(cat) of the V214A, V214L, V219T, and M226A mutants was increased up to 1.8-fold, and that of the V214T, F215A, F215L, F215Y, L218I, and L218V mutants was decreased 3-10.5-fold. Based on analysis of these mutations we suggest that cholesterol enters P450 7A1 through the membrane, and Val-214, Phe-215, and Leu-218 are the residues located near the point of cholesterol entry. The results provide an understanding of both the P450 7A1-membrane interactions and the mechanism for substrate specificity.

Amino Acid Sequence↗

PubMed2001

Golgi-localizing, gamma-adaptin ear homology domain, ADP-ribosylation factor-binding (GGA) proteins interact with acidic dileucine sequences within the cytoplasmic domains of sorting receptors through their Vps27p/Hrs/STAM (VHS) domains.

GGA (Golgi-localizing, gamma-adaptin ear homology domain, ARF-binding) proteins are potential effectors of ADP-ribosylation factors, are associated with the trans-Golgi network (TGN), and are involved in protein transport from this compartment. By yeast two-hybrid screening and subsequent two-hybrid and pull-down analyses, we have shown that GGA proteins, through their VHS (Vps27p/Hrs/STAM) domains, interact with acidic dileucine sequences found in the cytoplasmic domains of TGN-localized sorting receptors such as sortilin and mannose 6-phosphate receptor. A mutational analysis has revealed that a leucine pair and a cluster of acidic residues adjacent to the pair are mainly responsible for the interaction. A chimeric receptor with the sortilin cytoplasmic domain localizes to the TGN, whereas the chimeric receptor with a mutation at the leucine pair or the acidic cluster is mislocalized to punctate structures reminiscent of early endosomes. These results indicate that GGA proteins regulate the localization to or exit from the TGN of the sorting receptors.

ADP-Ribosylation Factors↗

PubMed2001

Deafness due to degeneration of cochlear neurons in caspase-3-deficient mice.

Mice that lack caspase-3, which functions in apoptosis, were generated by gene targeting and shown to undergo hearing loss. The ABR threshold of the caspase-3(-/-) mice was significantly elevated compared to that of caspase-3(+/+) mice at 15 days of age and was progressively elevated further by 30 days. Distortion product otoacoustic emissions were not detectable in caspase-3(-/-) mice at 15 days of age. Caspase-3(-/-) mice exhibited marked degeneration of spiral ganglion neurons and a loss of inner and outer hair cells in the cochlea at 30 days of age, although no such changes were apparent at 15 days. The degenerating neurons manifested features, including cytoplasmic vacuolization, distinct from those characteristic of apoptosis. Spiral ganglion neurons and cochlear hair cells thus appear to require caspase-3 for survival but not for initial development. The mapping of both the human caspase-3 gene and the locus responsible for an autosomal dominant, nonsyndromic form of hearing loss (DFNA24) to chromosome 4q35 suggests that the caspase-3(-/-) mice may represent a model of this human condition.

Aging↗

PubMed2001

Allelotype analysis of gallbladder carcinoma associated with anomalous junction of pancreaticobiliary duct.

Anomalous junction of pancreaticobiliary duct (AJPBD) patients has an increased risk of gallbladder and bile duct carcinomas. However, the relevance of carcinoma with AJPBD is not fully clarified. We performed analysis of loss of heterozygosity (LOH) at p53 locus and immunohistochemistry of p53 and K-ras gene mutation in five cases of gallbladder carcinoma associated with AJPBD. LOH of p53 locus and overexpression of p53 were detected in two out of five (40%) and five out of five (100%), respectively, in the present study. K-ras gene mutation at codon 12 and 13 was not detected (0%, 0/5). These results suggest that aberrations of p53 are involved in carcinogenesis of gallbladder carcinoma associated with AJPBD. Next, in order to find the genetic events besides K-ras mutation and overexpression of mutant p53 in this disease, LOH analysis was performed using 72 microsatellite markers. High frequency of allelic loss (> 50%) was found on 2p (81.8%), 4p (50%), 4q (50%), 8q (60%), 9q (50%), 10p (50%), 14p (60%), 14q (50%), 16p (60%), 19p (50%), 21p (50%) and Xp (66.6%). The highest deletion regions on chromosome 2p24 (3/3, 100%), 14q22 (3/4, 75%) and 21q22 (3/4, 75%) were found. The present study suggests that gallbladder carcinoma associated with AJPBD has high frequent allelic loss and has two new regions which may harbor putative tumor suppressor genes.

Aged↗

PubMed2001

Co-operative regulation of the transcription of human dihydrodiol dehydrogenase (DD)4/aldo-keto reductase (AKR)1C4 gene by hepatocyte nuclear factor (HNF)-4alpha/gamma and HNF-1alpha.

Human dihydrodiol dehydrogenase (DD) 4/aldo-keto reductase (AKR) 1C4 is a major isoform of hepatic DD that oxidizes trans-dihydrodiols of polycyclic aromatic hydrocarbons to reactive and redox-active o-quinones and that reduces several ketone-containing drugs. To investigate the mechanism of transcriptional regulation of the human DD4 gene, the 5'-flanking region of the gene was fused to the luciferase gene. The results of luciferase assays using HepG2 cells and of 1,10-phenanthroline-copper footprinting indicated that two positive regulatory regions were located in regions from -701 to -684 and from -682 to -666. The former region contained a putative hepatocyte nuclear factor (HNF)-4 binding motif, and the latter region contained an HNF-1 consensus binding sequence. DNA fragments of the HNF-4 or HNF-1 motif gave a shifted band in a gel-shift assay with nuclear extracts from HepG2 cells. The formation of the DNA-protein complex was inhibited by the HNF-4 or HNF-1 motif of the alpha(1)-antitrypsin gene. A supershift assay using antibodies to human HNF-4alpha, HNF-4gamma and HNF-1alpha showed that HNF-4alpha and HNF-4gamma bound to the HNF-4 motif, and that HNF-1alpha interacted with the HNF-1 motif. Introduction of mutations into the HNF-4 or HNF-1 motif lowered the luciferase activity to 10 or 8% respectively of that seen with the intact human DD4 gene. These results indicate that HNF-4alpha, HNF-4gamma and HNF-1alpha regulate co-operatively the transcription of the human DD4 gene in HepG2 cells.

Alcohol Oxidoreductases↗

PubMed2001

Regulation of the cell cycle at the G1-S transition by proteolysis of cyclin E and p27Kip1.

The transition from G1 phase to S phase of the mammalian cell cycle is controlled by many positive and negative regulators, among which cyclin E and p27Kip1, respectively, undergo the most marked changes in concentration at this transition. The abundance of both cyclin E and p27Kip1 is regulated predominantly by posttranslational mechanisms, in particular by proteolysis mediated by the ubiquitin-proteasome pathway. Cyclin E and p27Kip1 each bind to and undergo polyubiquitination by the same ubiquitin ligase, known as SCF(Skp2). The degradation of cyclin E and p27Kip1 is greatly impaired in Skp2-deficient mice, resulting in intracellular accumulation of these proteins. In this article, recent progress in characterization of the molecular mechanisms that control the proteolysis of cyclin E and p27Kip1 is reviewed.

Animals↗

PubMed2001

Cu-metallothioneins (Cu(I)8-MTs) in LEC rat livers 13 weeks after birth still act as antioxidants.

Redox properties of metallothioneins (MTs) and Cu in the cytosol from Long-Evans Cinnamon (LEC) rat livers 13 weeks after birth were investigated. MTs from LEC rat livers contain 8 g atoms of Cu and 1 g atom of Zn per mole of protein (Cu(I)8-MTs). Titration of Cu(I)8-MTs with CuCl2 indicates that Cu(I)8-MTs were able to reduce further 2-g atoms of cupric ions per mole MTs as bound form. Hg2+-induced hydroxyl radical generation from Cu(I)8-MTs was demonstrated by ESR using the spin trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The intensity of DMPO-OH signal from Cu-loaded MTs was increased with the increasing number of Cu in MTs. The used cytosol fraction contained 1.37 mM total Cu and 5 mM DTNB titrable-SH groups has a potential to reduce 2 mM CuCl2. No ESR signal due to Cu2+ was also detected with LEC rat liver cytosol, whereas strong Cu2+ signal appeared by the addition of HgCl2. The rate constants for the reaction of Cu(I)8-MTs with superoxide and hydroxyl radicals were estimated to be 2 x 10(6) and > or = 10(12) M(-1)s(-1), respectively, from competition kinetics. Cu2+-catalyzed oxidation of DNA was strongly inhibited both in the presence of Cu-unsaturated MTs and GSH. The results suggest that Cu(I)8-MTs from LEC rat livers just before hepatitis still act as antioxidants.

Animals↗

PubMed2001

Addressing the stability of C-capped dipeptide efflux pump inhibitors that potentiate the activity of levofloxacin in Pseudomonas aeruginosa.

Synthetic optimization of a biologically labile class of dipeptides that function as efflux pump inhibitors to potentiate the antibacterial agent levofloxacin in Pseudomonas aeruginosa has led to the discovery of a related series of compounds that are completely stable in a variety of biological matrices. Other than the stability profile, the in vitro profile of the new series is essentially identical to that observed with the original one. A prototypical compound from the new series demonstrates potentiation in an in vivo model of infection.

Animals↗

PubMed2001

High serum IgG4 concentrations in patients with sclerosing pancreatitis.

BACKGROUND: Sclerosing pancreatitis is a unique form of pancreatitis that is characterized by irregular narrowing of the main pancreatic duct, lymphoplasmacytic inflammation of the pancreas, and hypergammaglobulinemia and that responds to glucocorticoid treatment. Preliminary studies suggested that serum IgG4 concentrations are elevated in this disease but not in other diseases of the pancreas or biliary tract. METHODS: We measured serum IgG4 concentrations using single radial immunodiffusion and an enzyme-linked immunosorbent assay in 20 patients with sclerosing pancreatitis, 20 age- and sex-matched normal subjects, and 154 patients with pancreatic cancer, ordinary chronic pancreatitis, primary biliary cirrhosis, primary sclerosing cholangitis, or Sjögren's syndrome. Serum concentrations of immune complexes and the IgG4 subclass of immune complexes were determined by means of an enzyme-linked immunosorbent assay with monoclonal rheumatoid factor. RESULTS: The median serum IgG4 concentration in the patients with sclerosing pancreatitis was 663 mg per deciliter (5th and 95th percentiles, 136 and 1150), as compared with 51 mg per deciliter (5th and 95th percentiles, 15 and 128) in normal subjects (P<0.001). The serum IgG4 concentrations in the other groups of patients were similar to those in the normal subjects. In patients with sclerosing pancreatitis, serum concentrations of immune complexes and the IgG4 subclass of immune complexes were significantly higher before glucocorticoid therapy than after four weeks of such therapy. Glucocorticoid therapy induced clinical remissions and significantly decreased serum concentrations of IgG4, immune complexes, and the IgG4 subclass of immune complexes. CONCLUSIONS: Patients with sclerosing pancreatitis have high serum IgG4 concentrations, providing a useful means of distinguishing this disorder from other diseases of the pancreas or biliary tract.

Adult↗

PubMed2001

Complete genome sequence of enterohemorrhagic Escherichia coli O157:H7 and genomic comparison with a laboratory strain K-12.

Escherichia coli O157:H7 is a major food-borne infectious pathogen that causes diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. Here we report the complete chromosome sequence of an O157:H7 strain isolated from the Sakai outbreak, and the results of genomic comparison with a benign laboratory strain, K-12 MG1655. The chromosome is 5.5 Mb in size, 859 Kb larger than that of K-12. We identified a 4.1-Mb sequence highly conserved between the two strains, which may represent the fundamental backbone of the E. coli chromosome. The remaining 1.4-Mb sequence comprises of O157:H7-specific sequences, most of which are horizontally transferred foreign DNAs. The predominant roles of bacteriophages in the emergence of O157:H7 is evident by the presence of 24 prophages and prophage-like elements that occupy more than half of the O157:H7-specific sequences. The O157:H7 chromosome encodes 1632 proteins and 20 tRNAs that are not present in K-12. Among these, at least 131 proteins are assumed to have virulence-related functions. Genome-wide codon usage analysis suggested that the O157:H7-specific tRNAs are involved in the efficient expression of the strain-specific genes. A complete set of the genes specific to O157:H7 presented here sheds new insight into the pathogenicity and the physiology of O157:H7, and will open a way to fully understand the molecular mechanisms underlying the O157:H7 infection.

Bacterial Proteins↗

PubMed2001

Characterization of a novel synGAP isoform, synGAP-beta.

We cloned a cDNA encoding a novel synGAP, synGAP-d (GenBank(TM) accession number ), from a rat brain cDNA library. The clone consisted of 4801 nucleotides with a coding sequence of 3501 nucleotides, encoded a protein consisting of 1166 amino acids with >99% homology with 1092 amino acid overlaps to synGAP, and contained a 13-nucleotide insertion to the previously reported synGAP mRNAs, which suggested that the clone was a splice variant of synGAP. We also found that there are at least seven variants in the 3' portion of the synGAP mRNA and that they encoded five different protein isoforms. The coding sequence of these C-terminal variants were classified into alpha1, alpha2, beta1, beta2, beta3, beta4, and gamma, and synGAP-d was classified as the beta1 form. The previously reported synGAPs (synGAP-a, -b, and -c and p135synGAP) can be classified as the alpha1 isoform. All isoforms were expressed specifically in the brain. Unexpectedly, the beta isoform, which lacks a C-terminal PSD-95-binding motif ((S/T)XV), was more restricted to the postsynaptic density fraction than the motif-containing alpha1 isoform. The beta isoform did not interact with PSD-95 but specifically interacted with a nonphosphorylated alpha subunit of Ca(2+)/calmodulin-dependent protein kinase II through its unique C-terminal tail.

Aging↗

PubMed2001

Regulation of the level of Vesl-1S/Homer-1a proteins by ubiquitin-proteasome proteolytic systems.

The vesl-1S/homer-1a gene is up-regulated during seizure and long term potentiation. Other members of the Vesl family, Vesl-1L, -2, and -3, are constitutively expressed in the brain. We examined the regulatory mechanisms governing the expression level of Vesl-1S protein, either an exogenously introduced one in COS7 or human embryonic kidney 293T cells or an endogenous one in rat brain neurons in cultures. In both cases, application of proteasome inhibitors increased the amount of Vesl-1S protein but not that of Vesl-1L, -2, or -3 protein. Deletion analyses revealed that the C-terminal 11-amino acid region was responsible for the proteolysis of Vesl-1S by proteasomes. Application of proteasome inhibitors promoted ubiquitination of Vesl-1S protein but not that of the Vesl-1S deletion mutant, which evaded proteasome-mediated degradation. These results indicate that ubiquitin-proteasome systems are involved in the regulation of the expression level of Vesl-1S protein.

Amino Acid Sequence↗

PubMed2001

Selective roles of retinoic acid receptor and retinoid x receptor in the suppression of apoptosis by all-trans-retinoic acid.

Retinoic acids exert profound effects on many biological processes including cell proliferation, differentiation, and morphogenesis. We previously reported that all-trans-retinoic acid (t-RA) protected mesangial cells from H(2)O(2)-triggered apoptosis by suppressing the activator protein 1 (AP-1) pathway. It was via inhibition of c-fos and c-jun expression and suppression of c-Jun N-terminal kinase (JNK) activation. In this report, we investigated the involvement of retinoic acid receptor (RAR) and retinoid X receptor (RXR) in the antiapoptotic effect of t-RA in H(2)O(2)-exposed cells. We found that pretreatment with RAR pan-antagonist (AGN193109) or RXR pan-antagonist (HX531) attenuated the antiapoptotic effect of t-RA. Similarly, transient transfection with a dominant-negative mutant of RAR or a dominant-negative RXR diminished the antiapoptotic effect of t-RA. Both RAR and RXR antagonists reversed the suppressive effect of t-RA on AP-1 activity. However, the roles of RAR and RXR in the suppression of AP-1 components by t-RA were found to be different. RAR antagonist reversed the suppressive effect of t-RA on both c-fos and c-jun, whereas RXR antagonist reversed the effect of t-RA on c-fos but not c-jun. Furthermore, suppression of JNK activation by t-RA was observed even in the presence of RAR and RXR antagonists. Consistently, suppression of JNK by t-RA was not affected by overexpression of either the dominant-negative RAR or the dominant-negative RXR. These data elucidated that the antiapoptotic effect of t-RA is mediated by both nuclear receptor-dependent and -independent mechanisms.

Animals↗