Biomedical subjects

Ayako Nakamura

Publications and source records attributed to Ayako Nakamura.

At least 19 recordsLinked to original sources

PubMed2006

Intranuclear immunolocalization of 14-3-3 protein isoforms in brains with spinocerebellar ataxia type 1.

Immunolocalization of 14-3-3 protein isoforms, one of the interacters with ataxin 1, was investigated in spinocerebellar ataxia type 1 (SCA 1) brains using isoform-specific antibodies. Samples from the pons and from the cerebellum of four SCA1 cases and three controls were studied. The intensity of the immunoreactivity (IR) and its subcellular topography were analyzed. In control subjects, granular immunoreactivity for an epitope common to all known isoforms of 14-3-3 proteins (14-3-3 COM) found in the cytoplasm of some pontine and dentate nucleus neurons was weak. It was observed in some Purkinje cells, while its intensity varied. Many nuclei of those neurons and Purkinje cells of SCA1 were intensely immunopositive for 14-3-3 COM, while it was less in their cytoplasm. Expanded polyglutamine epitope was colocalized to 14-3-3 COM epitope in some pontine neurons, sometimes accumulated in intranuclear inclusion-like structures. This findings support previous reports that 14-3-3 proteins stabilize mutant ataxin 1 in nucleus and possibly lead to neurodegeneration. However, nuclear localization of 14-3-3 proteins in SCA1 brains was dependent on its isoforms, i.e. pontine neurons intensely positive for beta, Purkinje cells for tau and dentate nucleus neurons for both, while all of those neurons were consistently positive for zeta isoform, although sigma isoform tended to be located in the cytoplasm. Nuclear accumulation and isoform- and region-dependent subcellular localizations of 14-3-3 proteins may be related to SCA1 pathology, which exhibits marked regional variability.

14-3-3 Proteins↗

PubMed2006

Degeneration of cardiac sympathetic nerve can occur in multiple system atrophy.

Decreased cardiac uptake of meta-iodobenzylguanidine (MIBG) on [(123)I] MIBG myocardial scintigraphy, a sensitive biological marker for Parkinson's disease (PD), is related to cardiac sympathetic denervation in patients with PD. A slight decrease in cardiac uptake of MIBG has also been reported in some patients with multiple system atrophy (MSA). However, the pathophysiological mechanism accounting for the slight decrease in MIBG uptake in MSA remains to be elucidated. For confirmation, we examined cardiac tissue and sympathetic ganglia from patients with MSA. We immunohistochemically examined each specimen of 15 patients with MSA together with 10 control subjects using antibodies against tyrosine hydroxylase (TH) and neurofilament (NF). The number of TH-immunoreactive nerve fibers in the epicardium was preserved in 8 of 15 patients with MSA as well as in 10 control subjects. The number of TH-immunoreactive, but not of NF-immunoreactive nerve fibers in the epicardium was mildly or moderately decreased in six patients with MSA, of whom four showed a decrease of TH immunoreactivity in the neuronal somata in the sympathetic ganglia. Moreover, TH- and NF-immunoreactive nerve fibers almost entirely disappeared in the heart of one patient with MSA, in whom Lewy body pathology was present in the sympathetic ganglia. These findings suggest that mild degeneration of the cardiac sympathetic nerve can occur in MSA which is closely related to the pathological change of neurons in the sympathetic ganglia, accounting for the slight decrease in cardiac uptake of MIBG. Moreover, concurrent Lewy body pathology in the sympathetic ganglia might accelerate cardiac sympathetic denervation even in MSA.

Adrenergic Fibers↗

PubMed2006

Effects of halogenated WNA derivatives on sequence dependency for expansion of recognition sequences in non-natural-type triplexes.

Triplex-forming oligonucleotides (TFOs) are sequence-specific DNA-binding agents, but their target duplexes are limited to homopurine/homopyrimidine sequences because of interruption of the pyrimidines bases in the purine region. This problem has not been fully solved despite a wide variety of studies. Recently, we have developed a bicyclic system as a novel scaffold for nucleoside analogues (WNA, W-shaped nucleoside analogues) and determined two useful compounds, WNA-betaT (2) and WNA-betaC (5), for highly stable and selective triplex formation at a TA and a CG interrupting site, respectively. However, subsequent investigations have shown that the triplex formation using WNA is dependent on the neighboring bases of the TFOs. In this study, we have synthesized new WNA derivatives having halogenated recognition bases or benzene rings and evaluated the effects of the modifications on the triplex stability as well as selectivity. It has been found that the WNA-betaT analogues holding 5-halogenated pyrimidine bases (WNA-beta(Br)U (3) and WNA-beta(F)U (4)) exhibit high CG-selectivity. On the other hand, the WNA-betaT derivatives having the bromo-substituted benzene ring (mBr-WNA-betaT (10) and oBr-WNA-betaT (11)) have shown high selectivity to a TA interrupting site with high stability in the sequences to which the original WNA-betaT do not bind. Thus, sequence-dependency has been overcome by the sequence-dependent use of WNA-betaT, mBr-WNA-betaT, and oBr-WNA-betaT.

Base Sequence↗

PubMed2006

The role of OsBRI1 and its homologous genes, OsBRL1 and OsBRL3, in rice.

Since first identifying two alleles of a rice (Oryza sativa) brassinosteroid (BR)-insensitive mutant, d61, that were also defective in an orthologous gene in Arabidopsis (Arabidopsis thaliana) BRASSINOSTEROID INSENSITIVE1 (BRI1), we have isolated eight additional alleles, including null mutations, of the rice BRI1 gene OsBRI1. The most severe mutant, d61-4, exhibited severe dwarfism and twisted leaves, although pattern formation and differentiation were normal. This severe shoot phenotype was caused mainly by a defect in cell elongation and the disturbance of cell division after the determination of cell fate. In contrast to its severe shoot phenotype, the d61-4 mutant had a mild root phenotype. Concomitantly, the accumulation of castasterone, the active BR in rice, was up to 30-fold greater in the shoots, while only 1.5-fold greater in the roots. The homologous genes for OsBRI1, OsBRL1 and OsBRL3, were highly expressed in roots but weakly expressed in shoots, and their expression was higher in d61-4 than in the wild type. Based on these observations, we conclude that OsBRI1 is not essential for pattern formation or organ initiation, but is involved in organ development through controlling cell division and elongation. In addition, OsBRL1 and OsBRL3 are at least partly involved in BR perception in the roots.

Alleles↗

PubMed2006

Arabidopsis Aux/IAA genes are involved in brassinosteroid-mediated growth responses in a manner dependent on organ type.

We examined whether auxin/indole-3-acetic acid (Aux/IAA) proteins, which are key players in auxin-signal transduction, are involved in brassinosteroid (BR) responses. iaa7/axr2-1 and iaa17/axr3-3 mutants showed aberrant BR sensitivity and aberrant BR-induced gene expression in an organ-dependent manner. Two auxin inhibitors were tested in terms of BR responses. Yokonolide B inhibited BR responses, whereas p-chlorophenoxyisobutyric acid did not inhibit BR responses. DNA microarray analysis revealed that 108 genes were up-regulated, while only eight genes were down-regulated in iaa7. Among the genes that were up- or down-regulated in axr2, 22% were brassinolide-inducible genes, 20% were auxin-inducible genes, and the majority were sensitive neither to BR nor to auxin. An inhibitor of BR biosynthesis, brassinazole, inhibited auxin induction of the DR5-GUS gene, which consists of a synthetic auxin-response element, a minimum promoter, and a beta-glucuronidase. These results suggest that Aux/IAA proteins function in auxin- and BR-signaling pathways, and that IAA proteins function as the signaling components modulating BR sensitivity in a manner dependent on organ type.

Arabidopsis↗

PubMed2006

Production and characterization of auxin-insensitive rice by overexpression of a mutagenized rice IAA protein.

Since auxin was first isolated and characterized as a plant hormone, the underlying molecular mechanism of auxin signaling has been elucidated primarily in dicot plants represented by Arabidopsis. In monocot plants, the molecular mechanism of auxin signaling has remained unclear, despite various physiological experiments. To understand the function and mechanism of auxin signaling in rice (Oryza sativa), we focused on the IAA gene, a well-studied gene in Arabidopsis that serves as a negative regulator of auxin signaling. We found 24 IAA gene family members in the rice genome. OsIAA3 is one of these family members whose expression is rapidly increased in response to auxin. We produced transgenic rice harboring mOsIAA3-GR, which can overproduce mutant OsIAA3 protein containing an amino acid change in domain II to cause a gain-of-function phenotype, by treatment with dexamethasone. The transgenic rice was insensitive to auxin and gravitropic stimuli, and exhibited short leaf blades, reduced crown root formation, and abnormal leaf formation. These results suggest that, in rice, auxin is important for development and its signaling is mediated by IAA genes.

Amidohydrolases↗

PubMed2006

[Isolation rate and antifungal drug susceptibility of yeast like fungi isolated from blood or vascular catheter].

Increased resistance of strains to antifungal drugs has gained increasing attention. We studied the status of fungal isolation from blood and vascular catheters at Juntendo University Hospital from 1994 to 2002. The major fungi isolated were Candida albicans, Candida parapsilosis, Candida glabrata and Candida trophicalis, or 86% yeast-like fungi. Isolation of these fungi from vascular catheters is increasing. The effectiveness of 6 anti-fungal agents against 116 yeast-like fungi was measured by microdilution. In antifungal activity of micafungin (MCFG), MIC90, was < or = 0.03 g/mL for C. albicans, C. glabrata and C. tropicalis. MCFG showed the strongest antifungal activity among the drugs tested for above Candida spp.. Five of 37 strains of C. albicans were resistant to fluconazole (FLCZ) showing MIC > or =64 g/mL. These strains were also resistant to itraconazole (ITCZ) but MICs of MGFG, flurocytosine (5-FC) and amphotericin B (AMPH-B). Two of 38 strains of C. parapsilosis are resistant to flurocytosine (5-FC) showing MIC > or =64 g/mL. There is no resistant strain of fungi (yeast-like organisms) tested against AMPH-B. Six patients from whom resistant fungi were isolated from blood and vascular catheters have severe diseases and/or are have just undergone a major surgical operation. These results indicate that it is vital for deep mycosis to start early treatment with appropriate drugs selected based on rapid detection and identification of organisms and the drug susceptibility of organisms.

Antifungal Agents↗

PubMed2005

Enhanced expression of aquaporin 4 in human brain with inflammatory diseases.

Aquaporin 4 (AQP4), one of the water channel proteins on the plasma membrane of astrocytes, is up-regulated in various conditions with brain edema. Possible participation of AQP4 in various inflammatory lesions, more or less associated with edema, was examined in human autopsied brains. Immunohistochemistry was used to investigate AQP4 expression in autopsied brains with multiple sclerosis (MS), human immunodeficiency virus encephalitis (HIVE) or progressive multifocal leukoencephalopathy (PML). The cellular localization of AQP4 and its relation to inflammatory lesions were then examined with double-labeling immunohistochemistry. AQP4 immunoreactivity (IR) was restricted to astrocytes and localized to their entire processes, including their endfeet facing the abluminal surface of capillaries. In MS brains, AQP4-positive astrocytes were more abundant at the periphery of plaques than in their center, as seen in ischemic foci. Quantification of fluorescent signal demonstrated that AQP4 IR was greatly increased around plaques relative to that in unaffected area. Although the white matter was severely involved in HIVE and PML, AQP4-positive astrocytes were rare in the white matter even around perivascular active inflammatory foci. They were abundant in the gray matter and most prominent in the boundary between the gray and white matter, without apparent relation to inflammatory foci. Some bizarre astrocytes in PML exhibited AQP4 IR. Up-regulation of AQP4 was consistently found in astrocytes in various inflammatory lesions. However, the distribution of AQP4-positve astrocytes differed markedly according to disease and was not necessarily related to brain edema, indicating that functions and regulation of AQP4 in human brains are multiple.

AIDS Dementia Complex↗

PubMed2005

Progressive accumulation of ubiquitin and disappearance of alpha-synuclein epitope in multiple system atrophy-associated glial cytoplasmic inclusions: triple fluorescence study combined with Gallyas-Braak method.

Alpha-synuclein (alphaS) and ubiquitin (Ub) are shared constituents of glial cytoplasmic inclusions (GCIs) and Lewy bodies (LBs), both composed of fibrillary structures. Staining profiles of GCIs were investigated with triple immunofluorescence involving immunostaining for alphaS and Ub, both amplified with catalyzed reporter deposition, and a fluorochrome, thiazin red (TR) that has an affinity to fibrillary structures. After observation for the triple-fluorescent images, the sections were subsequently stained with the Gallyas-Braak method. Sections of putamen, cerebellar white matter and motor cortex from patients suffering from multiple system atrophy (MSA) with varying duration of the disease (4-15 years) were quantified for these staining profiles of Gallyas-positive GCIs. Although most of GCIs were positive for Ub and variably positive for alphaS, they were consistently negative for TR. The result was opposite in LBs in Lewy body disease with variable affinity to TR, suggesting that the construction of GCIs is different from that of LBs. These four staining features (alphaS, Ub, TR and Gallyas) alone failed to exhibit apparent correlation with disease duration, lesion site or severity of degeneration as reported previously. The fraction of alphaS-negative and Ub-positive GCIs, however, linearly increased along the disease progression, while that of alphaS-positive and Ub-negative GCIs decreased in contrast. This reciprocal change suggests that alphaS immunoreactivity in GCIs is being replaced by Ub immunoreactivity during the disease progression, which resulted in the ultimate predominance of alphaS-negative and Ub-positive GCIs in the most advanced case. Interestingly, this predominance of alphaS-negative and Ub-positive GCIs was a feature of motor cortex, where degeneration usually remains mild in spite of robust appearance of Gallyas-positive GCIs. Another fraction, alphaS-positive and Ub-positive GCIs were frequent in cerebellar white matter, suggesting that GCI evolution is heterogeneous and dependent also on area examined. Progressive accumulation of Ub with concomitant disappearance of alphaS epitope and their colocalization, partly shared with LBs, may represent a process of GCI formation, possibly linked to an aspect of degeneration in MSA.

Aged↗

PubMed2005

Silver stainings distinguish Lewy bodies and glial cytoplasmic inclusions: comparison between Gallyas-Braak and Campbell-Switzer methods.

Lewy bodies (LBs) of idiopathic Parkinson's disease and glial cytoplasmic inclusions (GCIs) of multiple system atrophy are pathological deposits both composed of phosphorylated alpha-synuclein woven into different filaments. Although both LBs and GCIs are considered to be hallmarks for each independent synucleinopathy, until now they could not be clearly distinguished on the basis of their biochemical or immunohistochemical features. We have examined possible differences in their argyrophilic features and their relation to synuclein-like or ubiquitin-like immunoreactivity (IR). Pairs of mirror sections from different brain areas were triple-fluorolabeled with an anti-alpha-synuclein antibody, an anti-ubiquitin antibody and thiazin red (TR), a fluorochrome that labels fibrillary structures such as Lewy bodies or neurofibrillary tangles. One of the paired sections was subsequently stained using the Campbell-Switzer method (CS), and the other by the Gallyas-Braak method (GB). By comparing of the same microscopic field on the paired fluorolabeled sections, subsequently silver-stained with either CS or GB, five different profiles of each structure could be determined: alpha-synuclein-like IR, ubiquitin-like IR, affinity to TR, argyrophilia with CS or GB. GCIs exhibited argyrophilia with both CS and GB but lacked affinity to TR. In contrast, LBs exhibited argyrophilia with CS but not with GB and some affinity to TR. These disease-specific profiles of argyrophilia were consistent, and were not influenced by areas or cases examined. Although immunohistochemical features of LBs and GCIs were similar in exhibiting IR for alpha-synuclein and ubiquitin, the contrast in their argyrophilic profiles may indicate possible differences in the molecular composition or conformation of alpha-synuclein. Even though these empirical differences still remain to be explained, awareness of this clear distinction is potentially of diagnostic and pathological relevance.

Aged↗

PubMed2005

Argyrophilic grains are not always argyrophilic--distinction from neurofibrillary tangles of diffuse neurofibrillary tangles with calcification revealed by comparison between Gallyas and Campbell-Switzer methods.

Silver staining profiles of argyrophilic grains (AGs) and of neurofibrillary tangles (NFTs) of diffuse neurofibrillary tangles with calcification (DNTC, collectively as DNTC-NFTs) were examined for their relation to tau- and ubiquitin-like immunoreactivity (IR). Pairs of mirror sections were triple-fluorolabeled with an anti-PHF tau (AT8) antibody, an anti-ubiquitin antibody and thiazin red (TR), a fluorochrome that identifies fibrillary structures such as NFTs of Alzheimer's disease (AD). One of the paired sections was subsequently stained with Gallyas method (GAL), and the other with Campbell-Switzer method (CS). Comparison of the same microscopic field on the paired fluorolabeled sections, subsequently silver-stained with either GAL or CS enabled the determination of five different profiles of each structure: AT8-IR, ubiquitin-like-IR, affinity to TR, argyrophilia with GAL or CS staining. AGs, mainly composed of four-repeat (4R) tau, were argyrophilic with GAL but not with CS, and their affinity to TR and ubiquitin-like-IR was not intense. This staining profile of AGs is identical with those of tau-positive structures in the cortex of progressive supranuclear palsy/corticobasal degeneration, both composed of 4R tau. This selective affinity of AGs to GAL is in sharp contrast with Pick bodies, composed of three-repeat (3R) tau, that are positive for CS but not for GAL, as we reported previously. This contrast is explainable if the argyrophilia with CS is related to deposits containing 3R tau, while that with GAL is linked to those containing 4R tau. Indeed, DNTC-NFTs, that contain both 3R and 4R tau, were argyrophilic with CS and GAL, and their affinity to TR and ubiquitin-like-IR were consistent, as we reported previously for NFTs of AD and of Down's syndrome, both similarly composed of 3R and 4R tau. Taken together, differences in molecular composition of tau protein in these deposits are linked to their argyrophilic properties dependent on the staining method in these sporadic tauopathies. Although explanations for these empirical differences are not yet available, awareness of this clear distinction is potentially of diagnostic and pathological significance.

Aged↗

PubMed2005

Transient abundance of presenilin 1 fragments/nicastrin complex associated with synaptogenesis during development in rat cerebellum.

Immunolocalization and expression of endogenous nicastrin (NCT) and presenilin 1 (PS1) fragments during postnatal development of rat cerebellum were investigated with fragment-specific antibodies. Immunoblotting for NCT revealed the expected mature and immature species, which gradually declined during development. In contrast, the expression of PS1 N-terminal fragment exhibited a peak at postnatal day 14 (P14) and declined thereafter. This chronological change was similarly observed with PS1 C-terminal fragment. Immunoprecipitation of NCT indicated its physical association with PS1 fragments. Colocalization of these molecules to the endoplasmic reticulum in cerebellar Purkinje cells indicates that they are organized into a complex in developing neurons. In addition, active sites of synaptogenesis, the base of the external granular layer and glomeruli, contained PS1 fragments and smaller amount of NCT. Isolated synaptic fraction contained both PS1 and NCT, suggesting their functional association within synapses. Transient abundance of NCT and PS1 fragments as a complex, when (P14) and where synaptogenesis is active, is consistent with intracellular trafficking of this complex in developing neurons and suggests its role as gamma-secretase in synaptogenesis.

Aging↗

PubMed2005

Silver staining profiles distinguish Pick bodies from neurofibrillary tangles of Alzheimer type: comparison between Gallyas and Campbell-Switzer methods.

Silver staining profiles of Pick bodies (PBs) and their relation to tau-like immunoreactivity were examined on hippocampal sections and compared with those of neurofibrillary tangles of Alzheimer type (NFTs). Pairs of mirror sections were double-fluorolabeled with an anti-paired helical filament tau (AT8) antibody and thiazin red (TR), a fluorochrome that identifies fibrillary structures such as NFTs. One of the paired sections was subsequently stained using the Gallyas method (GAL), and the other using the Campbell-Switzer method (CS). By comparison of the same microscopic field on fluorolabeled sections and on both silver-stained paired sections, four different profiles of each structure could be distinguished: AT8 immunoreactivity, affinity to TR, argyrophilia with GAL or CS staining. PBs, containing mainly three-repeat (3R) tau, were positive for CS but not for GAL and its affinity to TR was, at most, weak. This selective affinity of PBs to CS is in sharp contrast with tau-positive structures of corticobasal degeneration/progressive supranuclear palsy, which are positive for GAL but not for CS, as we reported previously. This contrast is explainable if the argyrophilia with CS is related to deposits containing 3R tau, while that with GAL is linked to those containing four-repeat (4R) tau. Indeed, NFTs, containing both 3R and 4R tau, are positive for both CS and GAL, as expected. Taken together, differences in molecular composition of tau protein in these deposits are linked to their argyrophilic properties that are dependent on the staining method. Although explanations for these empirical differences are not yet available, awareness of this clear distinction is potentially of diagnostic and pathological relevance.

Aged↗

PubMed2005

Effects of heat stress on the redox status in the oviduct and early embryonic development in mice.

This study examined the association between redox status in the oviduct and early embryonic death in heat-stressed mice. In Experiment 1, non-pregnant mice were heat-stressed at 35 C with 60% relative humidity for 12, 24, or 36 h, and the maternal redox status was verified by measuring the levels of reactive oxygen species (ROS) and free radical scavenging activity (FRSA) in the oviduct, and thiobarbituric acid reactive substances (TBARS) and glutathione peroxidase (GSH-Px) activity in the liver. In Experiment 2, zygotes were collected from mice heat-stressed for 12 h on the day of pregnancy, and their developmental abilities were assessed in vitro, along with the intensity of DNA damage at the 2-cell stage. The TBARS value and GSH-Px activity in the liver, and ROS level in the oviduct were significantly higher in heat-stressed mice, and this increase appeared to depend on the duration of the heat stress. Maternal heat stress significantly reduced the percentage of zygotes that developed to the morula and blastocyst and the total cell number in the blastocyst. In addition, DNA damage at the 2-cell stage was significantly higher in maternally heat-stressed embryos. These results suggest that heat stress induces systemic changes in redox status in the maternal body, and the resultant increase in oxidative stress in the oviduct is possibly involved in heat stress-induced early embryonic death .

Animals↗

PubMed2005

Expansion of triplex recognition codes by the use of novel bicyclic nucleoside derivatives (WNA).

Recently, we have developed new base analogs (WNA) and demonstrated that WNA-[see text];T with thymine and WNA-[see text];C with cytosine stabilize n on-natural antiparallel triplexes with a TA or CG interrupting site, respectively. However, limitations in recognizable sequences with the WNA-containing TFO were also found. The objective of this study is to search better WNA analogs for expansion of triplex recognition codes to general duplex sequences. In this study, we designed new WNA analogs by systematic modification of the aromatic part and the recognition part. The new WNA analogs with the benzene ring substituted with bromide or cyanide have determined for selective stabilization of triplexes at a TA interrupting site, and general formation of triplexes having a TA interrupting site has been achieved.

Base Composition↗

PubMed2005

Modification of the aromatic ring of the WNA analogues for expansion of the triplex recognition codes.

Triplex-forming oligonucleotides (TFOs) are powerful tools for genomic research. The most stable triplex is formed by the interaction between TFOs and homopurine/homopyrimidine sequences in the target duplex, but the triplex DNA is hampered by one pyrimidine base in the homopurine tract. Previously, we developed novel nucleoside analogues (WNA: W-shaped nucleoside analogues) to recognize pyrimidine/purine inversion sites (TA or CG interrupting sites) and determined two useful WNA analogues, WNA-betaT and WNA-betaC. However, subsequent study showed that the triplex formation using the WNA analogues was dependent on its neighbouring bases of the TFOs. In this study, the new WNA analogues having a different aromatic ring were synthesized to evaluate effects on sequence dependency. It has been found that o-bromo, m-bromo-, and p-cyano-substituted WNA-betaT derivatives are selective to a TA interrupting site to form triplexes with high stability in the sequences where original WNA-betaT could not recognize.

DNA↗

PubMed2005

Output of radiopharmaceutical nuclides of known injected doses from a municipal sewage treatment system.

The concentrations of (99m)Tc, (123)I, (67)Ga, and (201)Tl of the discharge water and the sewage sludge from Kurume Central Sewage Treatment Plant were determined once a week for 2 mo. The radiopharmaceutical doses injected into the patients for all of four hospitals that use nuclear medicines in the service area of the plant were also surveyed. Approximately 1.5% of the (99m)Tc, 1.5% of the (123)I, 4.3% of the (67)Ga, and 0.41% of the (201)Tl of the injected doses were detected in the discharged water from the plant. The behavior of these radionuclides in the sewage treatment system was analyzed using a compartment model. The analyses suggest that the average residence times in storage tanks and drainage pipes before entering the plant were 9.5 h for (99m)Tc, 81 h for (123)I, 120 h for (67)Ga, and 480 h for (201)Tl.

Gallium Radioisotopes↗

PubMed2005

DNA polymerase lambda directly binds to proliferating cell nuclear antigen through its confined C-terminal region.

DNA polymerase lambda (Pol lambda) was recently identified as a new member of the family X of DNA polymerases. Here, we show that Pol lambda directly binds to proliferating cell nuclear antigen (PCNA), an auxiliary protein for DNA replication and repair enzymes, both in vitro and in vivo. A pull-down assay using deletion mutants of Pol lambda showed that the confined C-terminal region of Pol lambda directly binds to PCNA. Furthermore, a synthetic peptide of 20-mers derived from the C-terminal region of Pol lambda competes with full-length Pol lambda for binding to PCNA. The residues between amino acids 518 and 537 of Pol lambda are required for binding to PCNA, and are different from the consensus PCNA interacting motif (PIM). Pol lambda associates with PCNA in vivo by immunoprecipitation analysis and EGFP-tagged Pol lambda co-localizes with PCNA as spots within a nucleus using fluorescent microscopy. Through direct binding, PCNA suppressed the distributive nucleotidyltransferase activity of Pol lambda. Pol micro, which also belongs to the family X of DNA polymerases, binds to PCNA by a pivotal amino acid residue.

Binding Sites↗