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Biomedical subjects

K Tamai

Publications and source records attributed to K Tamai.

At least 37 records · Page 2Linked to original sources
PubMed2001

Terminal deoxynucleotidyltransferase directly interacts with a novel nuclear protein that is homologous to p65.

BACKGROUND: Terminal deoxynucleotidyltransferase (TdT) is a DNA polymerase that enhances Ig and TcR gene diversity in the N region in B- and T-cells. TdT is found as a member of a large protein complex in the lysate of the thymocytes. To elucidate the molecular mechanism of the synthesis of the N region, we first attempted to isolate the genes with products that are interacting directly with TdT. RESULTS: Using a yeast two-hybrid system, we isolated a cDNA clone encoding a novel nuclear protein that interacts with TdT. This protein was designated as TdT interacting factor 1 (TdIF1). TdIF1 has a high degree of homology to the transcription factor p65, which belongs to the nuclear receptor superfamily. TdIF1 contains HMG-I and HMG-Y DNA binding domains (AT-hooks) and can bind to single- and double-stranded DNA. TdT and TdIF1 were co-eluted at position 232 kDa by gel filtration of MOLT4 lysate. TdIF1 can enhance TdT activity fourfold in vitro assay system using oligo(dT)16 as primers. CONCLUSIONS: TdIF1 binds directly to TdT, both in vitro and in vivo. TdIF1 and TdT exist as the members of a 232 kDa protein complex. TdIF1 can enhance TdT activity maximum fourfold in vitro assay system, suggesting that it positively regulates the synthesis of the N region during V(D)J recombination in the Ig and TcR genes.

Amino Acid Sequence↗
PubMed2001

Terminal deoxynucleotidyltransferase is negatively regulated by direct interaction with proliferating cell nuclear antigen.

BACKGROUND: The repertoires of Ig and TcR are generated by a combinatorial rearrangement of variable (V), diversity (D), and joining (J) segments (V(D)J recombination) in B- and T-cells. Terminal deoxynucleotidyltransferase (TdT) adds extra nucleotides (N nucleotides) at the junctions of the gene segments to enhance the Ig and TcR genes diversity. Using an anti-TdT antibody column, TdT has been purified as a member of a megadalton protein complex from rat thymus. The N region would be synthesized with the large protein complex. RESULTS: The cDNAs for proliferating cell nuclear antigen (PCNA) were isolated by yeast two-hybrid screening as the gene products which directly interacted with TdT. The interaction between PCNA and TdT was confirmed by co-immunoprecipitation, both in vitro and in vivo. TdT binds directly to a PCNA trimer, as shown by gel filtration. TdT interacts with PCNA in its DNA polymerization domain (DPD), but not in its BRCA-1 C-terminal (BRCT) domain. TdT activity was reduced to 17% of the maximum value by TdT/PCNA complex formation. CONCLUSION: TdT interacts directly with PCNA through its DPD. A functional consequence of this interaction is the negative regulation of TdT activity. These findings suggest that TdT catalyses the addition of N nucleotides under the negative control of PCNA during V(D)J recombination.

Binding Sites↗
PubMed2001

Hypercholesterolemia induces leukocyte entrapment in the retinal microcirculation of rats.

PURPOSE: To evaluate leukocyte dynamics in the retinal microcirculation of hypercholesterolemic rats in vivo using acridine orange digital fluorography. METHODS: 18 male pigmented rats were divided into 3 groups according to their diet; (A) control diet (0.1% cholesterol) for 8 weeks, (B) control diet for an initial 4 weeks followed by a high-cholesterol (HC) diet (5% cholesterol) for another 4 weeks and (C) HC diet for 8 weeks. Leukocyte dynamics were observed with acridine orange digital fluorography. The diameter of major retinal vessels and the number of leukocytes trapped in the retina were evaluated quantitatively. RESULTS: Both groups B and C showed approximately 4-fold higher serum cholesterol levels than in group A. The diameters of retinal arteries and veins were not significantly different among the three groups. Also, no rolling leukocytes were observed in any of the groups. In the retinal microcirculation at 30 minutes after acridine orange injection, the density of trapped leukocytes in both groups B and C was significantly greater than in group A (23.19 +/- 2.13, 28.56 +/- 1.96 and 13.67 +/- 1.45 cells/mm(2), respectively, p < 0.01). Furthermore, group C exhibited a significantly greater number of trapped leukocytes than in group B (p < 0.01). CONCLUSION: Enhanced leukocyte entrapment in the retinal microcirculation was induced in the early stage of hypercholesterolemia. Entrapment was increased as the period of hypercholesterolemia was prolonged. Leukocyte accumulation in the retina may be an early vascular dysfunction leading to arteriosclerosis in hypercholesterolemia.

Acridine Orange↗
PubMed2001

Orientation and osteoarthritis of the lumbar facet joint.

Several studies have shown an association between sagittal orientation of the facet joint and degenerative spondylolisthesis. There is currently no information available on the association between orientation of the facet joint and osteoarthritis. This study examined the association between orientation and osteoarthritis of the lumbar facet joints. One hundred eleven consecutive patients underwent plain radiography and magnetic resonance imaging. These patients were divided into two groups: No Degenerative Spondylolisthesis Group (98 patients) and Degenerative Spondylolisthesis Group (13 patients). In the No Degenerative Spondylolisthesis Group, segments with higher grades of osteoarthritis showed more sagittal orientation of the facetjoints at the L3-L4 and L4-L5 levels. The facet joint was oriented significantly more sagittally in the Degenerative Spondylolisthesis Group than in the No Degenerative Spondylolisthesis Group at the L4-L5 and L5-S1 levels. The severity of facet joint osteoarthritis was significantly higher in the Degenerative Spondylolisthesis Group than in the No Degenerative Spondylolisthesis Group at the L3-L4, L4-L5, and L5-S1 levels. A significant association was found between sagittal orientation and osteoarthritis of the lumbar facet joints, even in patients without degenerative spondylolisthesis. Facet joint osteoarthritis, rather than spondylolisthesis, is the pathoanatomic feature that is associated with sagittal orientation of the facet joints in patients with degenerative spondylolisthesis.

Aged↗
PubMed2001

Anterior dislocation of the subtalar joint: a case report.

We report a very rare case of anterior dislocation of the subtalar joint. Forceful supination of the foot and dorsiflexion of the ankle was considered the cause of the injury in this case. Closed reduction was successful for the talocalcaneal component of subtalar joint, although surgery was subsequently performed because of the residual subluxation of the midtarsal joint including the talonavicular component of subtalar joint and the associated fracture of the lateral process of the talus. Satisfactory results were shown at three-year follow-up.

Accidents, Traffic↗
PubMed2001

Analysis of calcium deposits in calcific periarthritis.

OBJECTIVE: To determine if hydroxyapatite (HAP), octacalcuim phosphate (OCP), or tricalcium phosphate (TCP) can be found in the calcium deposits in calcific periarthritis. METHODS: Thirty-six specimens from 34 patients who had acute inflammation and roentgenographically recognized calcification in soft tissue were analyzed. Twenty-three patients with calcific tendinitis in the shoulder and 11 with calcific periarthritis at other sites were included. We prepared 2 kinds of samples from each specimen; a dried sample (washed and dried calcific deposit), and a sample heated to 1,000 degrees C. All were analyzed by X-ray diffraction, Raman spectroscopy, infrared absorption spectroscopy, and X-ray fluorescence spectrometry for calcium and phosphorus molar ratio. Synthetic HAP was used as the control in each analysis. RESULTS: The X-ray diffraction patterns of all dried samples were similar to those of HAP and carbonate apatite. We found no diffraction patterns of OCP or TCP. However, an OH- group at 3570cm(-1) was observed with Raman spectroscopy for samples heated to 1,000 degrees C and synthetic HAP, but not for the dried samples. Infrared absorption spectroscopy also confirmed an OH- group for samples heated to 1,000 degrees C and synthetic HAP, and confirmed that dried samples contained carbonate. CONCLUSION: Calcium deposits are composed of carbonate apatite. HAP, OCP, and TCP were not identified in any deposits.

Adult↗
PubMed2001

[Clinical and MRI findings in childhood idiopathic intracranial hypertension].

We report here clinical and MRI findings of four children with idiopathic intracranial hypertension (IIH). Their chief complaint was headache. Three patients had papilloedema, and the other one showed the highest cerebrospinal fluid pressure (106 cm H2O) without papilloedema. In two cases, the symptoms disappeared after repeated lumbar punctures. At admission, MRI demonstrated empty sella in all the four cases. Regarding the optic nerves, there were more than two of the following three findings: distension of the perioptic subarachnoid space, vertical tortuosity and elongation of the optic nerve, and flattening of the posterior aspect of the globe. Follow-up MRI showed normalization of sella turtica and/or optic nerve findings in two of the three cases examined. MRI focusing on the optic nerves and pituitary gland may provide important clues for the diagnosis of IIH, especially those without papilloedema.

Adolescent↗
PubMed2001

[Duration of ACTH therapy for West syndrome].

We retrospectively studied 18 patients with West syndrome treated with ACTH according to Fukuyama's method. ACTH was given daily for the first 2 weeks, once every other day for the 3rd and 4th weeks, twice for the 5th and 6th weeks, and once a week for the 7th and 8th weeks. The patients were classified into two groups; group 1 (n = 9), in which Fukuyama's method was competed by a total of 27 injections; and group 2 (n = 9), in which Fukuyama's method was discontinued due to its adverse effects. In group 2, the duration of ACTH therapy was 15 days on average, and shorter than that in group 1. Between the two groups, there was no significant difference in the control rate of clinical seizures, and in the duration of therapy to attain seizure control (6 to 8 days on average). These results suggest that the tapering period is unnecessary after the cessation of spasms.

Adrenocorticotropic Hormone↗
PubMed2001

Role of root hairs and lateral roots in silicon uptake by rice.

The rice plant (Oryza sativa L. cv Oochikara) is known to be a Si accumulator, but the mechanism responsible for the high uptake of Si by the roots is not well understood. We investigated the role of root hairs and lateral roots in the Si uptake using two mutants of rice, one defective in the formation of root hairs (RH2) and another in that of lateral roots (RM109). Uptake experiments with nutrient solution during both a short term (up to 12 h) and relatively long term (26 d) showed that there was no significant difference in Si uptake between RH2 and the wild type (WT), whereas the Si uptake of RM109 was much less than that of WT. The number of silica bodies formed on the third leaf in RH2 was similar to that in WT, but the number of silica bodies in RM109 was only 40% of that in WT, when grown in soil amended with Si under flooded conditions. There was also no difference in the shoot Si concentration between WT and RH2 when grown in soil under upland conditions. Using a multi-compartment transport box, the Si uptake at the root tip (0-1 cm, without lateral roots and root hairs) was found to be similar in WT, RH2, and RM109. However, the Si uptake in the mature zone (1-4 cm from root tip) was significantly lower in RM109 than in WT, whereas no difference was found in Si uptake between WT and RH2. All these results clearly indicate that lateral roots contribute to the Si uptake in rice plant, whereas root hairs do not. Analysis of F(2) populations between RM109 and WT showed that Si uptake was correlated with the presence of lateral roots and that the gene controlling formation of lateral roots and Si uptake is a dominant gene.

Cell Surface Extensions↗
PubMed2000

Multiple Ras-dependent phosphorylation pathways regulate Myc protein stability.

Our recent work has shown that activation of the Ras/Raf/ERK pathway extends the half-life of the Myc protein and thus enhances the accumulation of Myc activity. We have extended these observations by investigating two N-terminal phosphorylation sites in Myc, Thr 58 and Ser 62, which are known to be regulated by mitogen stimulation. We now show that the phosphorylation of these two residues is critical for determining the stability of Myc. Phosphorylation of Ser 62 is required for Ras-induced stabilization of Myc, likely mediated through the action of ERK. Conversely, phosphorylation of Thr 58, likely mediated by GSK-3 but dependent on the prior phosphorylation of Ser 62, is associated with degradation of Myc. Further analysis demonstrates that the Ras-dependent PI-3K pathway is also critical for controlling Myc protein accumulation, likely through the control of GSK-3 activity. These observations thus define a synergistic role for multiple Ras-mediated phosphorylation pathways in the control of Myc protein accumulation during the initial stage of cell proliferation.

Amino Acid Sequence↗
PubMed2000

LDL-receptor-related proteins in Wnt signal transduction.

The Wnt family of secreted signalling molecules are essential in embryo development and tumour formation. The Frizzled (Fz) family of serpentine receptors function as Wnt receptors, but how Fz proteins transduce signalling is not understood. In Drosophila, arrow phenocopies the wingless (DWnt-1) phenotype, and encodes a transmembrane protein that is homologous to two members of the mammalian low-density lipoprotein receptor (LDLR)-related protein (LRP) family, LRP5 and LRP6 (refs 12-15). Here we report that LRP6 functions as a co-receptor for Wnt signal transduction. In Xenopus embryos, LRP6 activated Wnt-Fz signalling, and induced Wnt responsive genes, dorsal axis duplication and neural crest formation. An LRP6 mutant lacking the carboxyl intracellular domain blocked signalling by Wnt or Wnt-Fz, but not by Dishevelled or beta-catenin, and inhibited neural crest development. The extracellular domain of LRP6 bound Wnt-1 and associated with Fz in a Wnt-dependent manner. Our results indicate that LRP6 may be a component of the Wnt receptor complex.

Animals↗
PubMed2000

p53AIP1, a potential mediator of p53-dependent apoptosis, and its regulation by Ser-46-phosphorylated p53.

Through direct cloning of p53 binding sequences from human genomic DNA, we have isolated a novel gene, designated p53AIP1 (p53-regulated Apoptosis-Inducing Protein 1), whose expression is inducible by wild-type p53. Ectopically expressed p53AIP1, which is localized within mitochondria, leads to apoptotic cell death through dissipation of mitochondrial A(psi)m. We have found that upon severe DNA damage, Ser-46 on p53 is phosphorylated and apoptosis is induced. In addition, substitution of Ser-46 inhibits the ability of p53 to induce apoptosis and selectively blocks expression of p53AIP1. Our results suggest that p53AIP1 is likely to play an important role in mediating p53-dependent apoptosis, and phosphorylation of Ser-46 regulates the transcriptional activation of this apoptosis-inducing gene.

Adenocarcinoma↗
PubMed2000

Human Cdc7-related kinase complex. In vitro phosphorylation of MCM by concerted actions of Cdks and Cdc7 and that of a criticial threonine residue of Cdc7 bY Cdks.

huCdc7 encodes a catalytic subunit for Saccharomyces cerevisae Cdc7-related kinase complex of human. ASK, whose expression is cell cycle-regulated, binds and activates huCdc7 kinase in a cell cycle-dependent manner (Kumagai, H., Sato, N., Yamada, M., Mahony, D. , Seghezzi, W., Lees, E., Arai, K., and Masai, H. (1999) Mol. Cell. Biol. 19, 5083-5095). We have expressed huCdc7 complexed with ASK regulatory subunit using the insect cell expression system. To facilitate purification of the kinase complex, glutathione S-transferase (GST) was fused to huCdc7 and GST-huCdc7-ASK complex was purified. GST-huCdc7 protein is inert as a kinase on its own, and phosphorylation absolutely depends on the presence of the ASK subunit. It autophosphorylates both subunits in vitro and phosphorylates a number of replication proteins to different extents. Among them, MCM2 protein, either in a free form or in a MCM2-4-6-7 complex, serves as an excellent substrate for huCdc7-ASK kinase complex in vitro. MCM4 and MCM6 are also phosphorylated by huCdc7 albeit to less extent. MCM2 and -4 in the MCM2-4-6-7 complex are phosphorylated by Cdks as well, and prior phosphorylation of the MCM2-4-6-7 complex by Cdks facilitates phosphorylation of MCM2 by huCdc7, suggesting collaboration between Cdks and Cdc7 in phosphorylation of MCM for initiation of S phase. huCdc7 and ASK proteins can also be phosphorylated by Cdks in vitro. Among four possible Cdk phosphorylation sites of huCdc7, replacement of Thr-376, corresponding to the activating threonine of Cdk, with alanine (T376A mutant) dramatically reduces kinase activity, indicative of kinase activation by phosphorylation of this residue. In vitro, Cdk2-Cyclin E, Cdk2-Cyclin A, and Cdc2-Cyclin B, but not Cdk4-Cyclin D1, phosphorylates the Thr-376 residue of huCdc7, suggesting possible regulation of huCdc7 by Cdks.

Antigens, Viral, Tumor↗
PubMed2000

Ataxia telangiectasia-mutated phosphorylates Chk2 in vivo and in vitro.

The protein kinase Chk2, the mammalian homolog of the budding yeast Rad53 and fission yeast Cds1 checkpoint kinases, is phosphorylated and activated in response to DNA damage by ionizing radiation (IR), UV irradiation, and replication blocks by hydroxyurea (HU). Phosphorylation and activation of Chk2 are ataxia telangiectasia-mutated (ATM) dependent in response to IR, whereas Chk2 phosphorylation is ATM-independent when cells are exposed to UV or HU. Here we show that in vitro, ATM phosphorylates the Ser-Gln/Thr-Gln (SQ/TQ) cluster domain (SCD) on Chk2, which contains seven SQ/TQ motifs, and Thr68 is the major in vitro phosphorylation site by ATM. ATM- and Rad3-related also phosphorylates Thr68 in addition to Thr26 and Ser50, which are not phosphorylated to a significant extent by ATM in vitro. In vivo, Thr68 is phosphorylated in an ATM-dependent manner in response to IR, but not in response to UV or HU. Substitution of Thr68 with Ala reduced the extent of phosphorylation and activation of Chk2 in response to IR, and mutation of all seven SQ/TQ motifs blocked all phosphorylation and activation of Chk2 after IR. These results suggest that in vivo, Chk2 is directly phosphorylated by ATM in response to IR and that Chk2 is regulated by phosphorylation of the SCD.

Amino Acid Sequence↗
PubMed2000

Chk1 is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint.

Chk1, an evolutionarily conserved protein kinase, has been implicated in cell cycle checkpoint control in lower eukaryotes. By gene disruption, we show that CHK1 deficiency results in a severe proliferation defect and death in embryonic stem (ES) cells, and peri-implantation embryonic lethality in mice. Through analysis of a conditional CHK1-deficient cell line, we demonstrate that ES cells lacking Chk1 have a defective G(2)/M DNA damage checkpoint in response to gamma-irradiation (IR). CHK1 heterozygosity modestly enhances the tumorigenesis phenotype of WNT-1 transgenic mice. We show that in human cells, Chk1 is phosphorylated on serine 345 (S345) in response to UV, IR, and hydroxyurea (HU). Overexpression of wild-type Atr enhances, whereas overexpression of the kinase-defective mutant Atr inhibits S345 phosphorylation of Chk1 induced by UV treatment. Taken together, these data indicate that Chk1 plays an essential role in the mammalian DNA damage checkpoint, embryonic development, and tumor suppression, and that Atr regulates Chk1.

Alleles↗
PubMed2000

The interspinous ligament of the lumbar spine. Magnetic resonance images and their clinical significance.

STUDY DESIGN: A preliminary study of magnetic resonance features of the interspinous ligament in degenerative lumbar spine. OBJECTIVES: To classify the magnetic resonance imaging features of the interspinous ligaments in relation to the patient's age, disc degeneration, and radiographic instability. Magnetic resonance imaging also was correlated with the histologic findings of the interspinous ligaments. SUMMARY OF BACKGROUND DATA: As reported, rupture of the interspinous ligament frequently is found in the degenerative lumbar spine. However, little information is available in the literature on imaging assessment of the interspinous ligament in degenerative lumbar disorders. METHODS: In this study, 24 interspinous ligaments at L1-L2 or L2-L3 from 15 patients with nondegenerated discs were selected to represent normal magnetic resonance features of the interspinous ligament, and 38 patients with the mean age of 49 years underwent functional radiography and magnetic resonance imaging. The magnetic resonance features of the interspinous ligament were classified into five categories according to their signal intensities: Type 1A (low intensity on T1- and T2-weighted images without hypertrophy of the spinal process); Type 1B (same signal pattern as in Type 1A with hypertrophy of spinal process); Type 2 (low intensity on T1- and high intensity on T2-weighted images); Type 3 (high intensity on T1-weighted images); and Type 4 (others). Seven patients with variable patterns of the interspinous ligament were selected to undergo histologic examinations. RESULTS: Of the interspinous ligaments considered normal, 80% were classified as Type 1A. There were 14 Type 1A, 30 Type 1B, 19 Type 2, 16 Type 3, and 20 Type 4 ligaments. The mean age and disc degeneration grade of the patients with the Type 1B ligaments was significantly higher. Instability was found to be associated with Type 2 interspinous ligaments (7 of 19), whereas instability rarely was noted in Types 1A (1 of 14) and 1B (1 of 30) ligaments. The histologic examination revealed that chondrometaplasia and necrotization of fiber bundle predominated in Type 1B, proliferation of cells and vascular invasion in Type 2, fatty degeneration in Type 3 ligaments. CONCLUSIONS: The magnetic resonance imaging characteristics may be helpful in assessing normal or pathologic changes in the interspinous ligaments.

Adolescent↗