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Biomedical subjects

K Tamai

Publications and source records attributed to K Tamai.

At least 91 records · Page 5Linked to original sources
PubMed1998

Activation of the ATM kinase by ionizing radiation and phosphorylation of p53.

The p53 tumor suppressor protein is activated and phosphorylated on serine-15 in response to various DNA damaging agents. The gene product mutated in ataxia telangiectasia, ATM, acts upstream of p53 in a signal transduction pathway initiated by ionizing radiation. Immunoprecipitated ATM had intrinsic protein kinase activity and phosphorylated p53 on serine-15 in a manganese-dependent manner. Ionizing radiation, but not ultraviolet radiation, rapidly enhanced this p53-directed kinase activity of endogenous ATM. These observations, along with the fact that phosphorylation of p53 on serine-15 in response to ionizing radiation is reduced in ataxia telangiectasia cells, suggest that ATM is a protein kinase that phosphorylates p53 in vivo.

Ataxia Telangiectasia Mutated Proteins↗
PubMed1998

The inhibitory effect of novel triterpenoid compounds, fomitellic acids, on DNA polymerase beta.

We previously found new triterpenoid compounds, designated fomitellic acid A and B, which selectively inhibit the activities of mammalian DNA polymerase alpha and beta in vitro. On DNA polymerase beta, the fomitellic acids acted by competing with both the substrate and the template primer, but on DNA polymerase alpha, they acted non-competitively. At least on DNA polymerase beta, the evidence suggests that each of the fomitellic acids bind to the active region competing with the substrate and/or template primer, and subsequently inhibits the catalytic activity. We therefore further investigated the enzyme-binding properties by using DNA polymerase beta and its proteolytic fragments. The 39 kDa enzyme was proteolytically separated into two fragments of the template-primer-binding domain (8 kDa) and the catalytic domain (31 kDa). The fomitellic acids bound tightly to the 8 kDa fragment, but not to the 31 kDa fragment. The immuno-precipitation by antibodies against the enzyme or each of the fragments also proved the binding. These results suggest that the fomitellic acid molecule competes with the template-primer molecule on its 8 kDa binding site, binds to the site, and the fomitellic acid molecule simultaneously disturbs the substrate incorporation into the template primer.

Animals↗
PubMed1998

Keratinocyte gene therapy for systemic diseases. Circulating interleukin 10 released from gene-transferred keratinocytes inhibits contact hypersensitivity at distant areas of the skin.

This study has examined the systemic effects of a circulating gene product, human interleukin 10 (IL-10), released from transduced keratinocytes. IL-10 is an anti-inflammatory cytokine which has an inhibitory effect on contact hypersensitivity (CHS). An expression vector (phIL-10) was constructed for human IL-10 and was injected into the dorsal skin of hairless rats. Local expression of IL-10 mRNA and protein was detected by reverse-transcriptase polymerase chain reaction and immunohistochemical staining, respectively. Enzyme-linked immunosorbent assay showed that the amount of IL-10 in the local keratinocytes and in the circulation increased with the dose of phIL-10 transferred. To determine whether circulating IL-10 could inhibit the effector phase of CHS at a distant area of the skin, various doses of phIL-10 were injected into the dorsal skin of sensitized rats before challenge on the ears. Our results showed that the degree of swelling of the ears of phIL-10- treated rats was significantly lower than that in the negative control animals. These results suggest that IL-10 released from transduced keratinocytes can enter the bloodstream and cause biological effects at distant areas of the skin. This study demonstrates that it may be possible to treat systemic disease using keratinocyte gene therapy.

Animals↗
PubMed1998

Different ocular abnormalities in individuals of a three-generation family caused by a new nonsense mutation in the PST domain of the PAX6 gene. Mutations in brief no. 189. Online.

PAX6 is a candidate gene for familial aniridia. We have carried out a mutational analysis of the PAX6 gene in a three-generation family from Germany, containing 5 individuals affected with ocular abnormalities. In all affected individuals, a heterozygous mutation was detected in the PAX6 gene, exchanging tyrosine 369 by a stop codon. The mutation is located in the 3' moiety of the PST domain, at the C terminus of the PAX6 protein. In the affected family members, the same heterozygous mutation leads to distinct phenotypes of varying severity. Most notably, no aniridia was observed in one of the family members carrying the mutation, although other ocular abnormalities (underdeveloped iris and cataracts) were present. We discuss the possibility that small C terminal truncations of the PAX6 protein might lead to less severe or more divergent phenotypes than trancations at internal positions.

Aniridia↗
PubMed1998

Localization of tyrosine-phosphorylated proteins in cultured mouse dorsal root ganglion neurons.

The present study, using confocal laser scanning microscopy and immunoelectron microscopy, examined the intracellular localization of tyrosine-phosphorylated proteins in cultured mouse dorsal root ganglion neurons with special reference to their growth cones. The growth cone is the specialized structure formed at the growing tip of the axon; characteristically highly motile with filopodia on the surface, it is responsible for the extension and guidance of the neurites to the appropriate targets during nerve regeneration. It has been suggested that protein-tyrosine phosphorylation plays an important role in the intracellular signal transduction that regulates the extension and motility of growth cones. By fluorescence immunocytochemistry, phosphotyrosine immunoreactivity was found in the growth cones and neurites. Some of the filopodia exhibited strong immunoreactivity at their tips. By immunoelectron microscopy, a large number of immunogold particles (gold particles conjugated to the secondary antibody) were seen to be distributed in the cytoplasm and some were observed on the plasma membrane in the growth cones, whereas in the neurites the density of immunogold particles was the same in the axoplasm as on the plasma membranes. These findings suggest that in the growth cones phosphotyrosines might mainly be involved in intracellular signaling for maintaining their high motility whereas in the neurites they might mostly be associated with the receptor proteins at the plasma membrane for adhesion as well as for growth of neurites. Thus, tyrosine phosphorylation might contribute to different functions for growth cones and neurites.

Animals↗
PubMed1998

AMY-1, a novel C-MYC binding protein that stimulates transcription activity of C-MYC.

BACKGROUND: The c-myc proto-oncogene has been suggested to play key roles in cell proliferation, differentiation, transformation and apoptosis. A variety of functions of C-MYC, the product of c-myc, are attributed to protein-protein interactions with various cellular factors including Max, YY1, p107, Bin1 and TBP. Max and YY1 bind to the C-terminal region of C-MYC, while p107, Bin1 and TBP bind to the N-terminal region covering myc boxes. The N-terminal region is involved in all the biological functions of C-MYC, and different proteins are therefore thought to interact with the N-terminal region of C-MYC to display different functions. RESULTS: We cloned two cDNAs which encode a novel C-MYC-binding protein of 11 kDa, designated AMY-1 (Associate of C-MYC). The two cDNAs, AMY-1L and AMY-1S, derived from alternative usage of polyadenylation signals, code for the same protein of 11 kDa. AMY-1 was bound via its C-terminal region to the N-terminal region of C-MYC (amino acids nos 58-148) corresponding to the transactivation domain. AMY-1 was localized in the cytoplasm in cells expressing c-myc at low levels, but in the nucleus in the cells of a high c-myc expression in transiently transfected cells. A similar difference in endogenous AMY-1 localization was observed during the cell cycle: AMY-1 translocated from cytoplasm to nucleus during the S phase when c-myc expression was increased. AMY-1 by itself did not recognize the E-box element, the MYC/Max binding sequence, nor did it transactivate via the element, but stimulated the activation of E-box-regulated transcription by MYC/Max. FISH analyses revealed that the amy-1 gene was located at 1p32.2-1p33 in human genome. CONCLUSIONS: AMY-1 is a 11 kDa protein which binds to the N-terminal region of C-MYC and stimulates the activation of E-box-dependent transcription by C-MYC. AMY-1, which mostly localizes in the cytoplasm, translocates into the nucleus in the S phase of the cell cycle upon an increase of c-myc expression, and may thus control the transcriptional activity of C-MYC.

Amino Acid Sequence↗
PubMed1998

Inhibitory effect of beta-thujaplicin on ultraviolet B-induced apoptosis in mouse keratinocytes.

Sunburn cells are thought to represent ultraviolet B-induced apoptotic keratinocytes. It has been demonstrated that enzymatic and nonenzymatic antioxidants effectively suppress sunburn cell formation, indicating that reactive oxygen species may play a role in the progression of ultraviolet B-induced apoptosis. Metallothionein, a cytosol protein, has antioxidant activity, and overexpression of metallothionein has been reported to reduce the number of sunburn cells in mouse skin. We have also demonstrated that overexpression of metallothionein inhibits ultraviolet B-induced DNA ladder formation in mouse keratinocytes. These findings support the hypothesis that cellular metallothionein may play an important role in the inhibition of ultraviolet B-induced apoptosis in keratinocytes through its antioxidant activity. In the present study, we investigated the effects of beta-thujaplicin, an extract from the woods of Thuja plicata D. Don. and Chamaecyparis obtuse, Sieb. et Zucc., on ultraviolet B-induced apoptosis in keratinocytes and on metallothionein induction. Topical application of beta-thujaplicin decreased the number of ultraviolet B-mediated sunburn cells and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling-positive cells in mouse ear skin. Incubation with beta-thujaplicin suppressed ultraviolet B-induced DNA ladder formation in cultured mouse keratinocytes. Histochemical analysis showed that topical application of beta-thujaplicin induced metallothionein protein in mouse skin. Northern analysis and western blotting revealed significant induction of metallothionein mRNA and metallothionein protein, respectively, in beta-thujaplicin-treated cultured mouse keratinocytes. These findings indicate that beta-thujaplicin inhibits ultraviolet B-induced apoptosis in keratinocytes and strongly suggest that the inhibitory mechanism is due to the antioxidant activity of metallothionein induced by the agent.

Animals↗
PubMed1998

Novel function of metallothionein in photoprotection: metallothionein-null mouse exhibits reduced tolerance against ultraviolet B injury in the skin.

We have shown previously that injection of cadmium chloride (Cd2+) depletes the number of ultraviolet B (UVB)-induced sunburn cells in the mouse skin in vivo, and that Cd2+ treatment enhances UVB resistance in cultured keratinocytes in vitro, indicating the photoprotective role of Cd2+-induced metallothioneins (MT) with antioxidant property against UVB injury; however, there has been no direct evidence for the role of MT in UV protection. To improve our understanding of MT in photoprotection, MT-null mouse deficient in its MT-1 and MT-2 genes was studied. Skin explants were preliminarily exposed to medium alone, Cd2+ and Cd2+ plus buthionine S,R-sulfoximine, an inhibitor of glutathione synthesis. We then compared the number of UVB-induced sunburn cells and apoptotic cells in the epidermis of MT-null mice with that of control mice using organ culture systems. The skin of MT-null mice developed a greater number of sunburn cells and apoptotic cells than did that of normal mice in all experimental conditions. These findings indicate that the skin of MT-null mouse is readily injured by UVB irradiation. MT-null mouse provided direct evidence of the photoprotective effect of cellular MT in the skin.

Animals↗
PubMed1998

The second-largest subunit of the mouse DNA polymerase alpha-primase complex facilitates both production and nuclear translocation of the catalytic subunit of DNA polymerase alpha.

DNA polymerase alpha-primase is a replication enzyme necessary for DNA replication in all eukaryotes examined so far. Mouse DNA polymerase alpha is made up of four subunits, the largest of which is the catalytic subunit with a molecular mass of 180 kDa (p180). This subunit exists as a tight complex with the second-largest subunit (p68), whose physiological role has remained unclear up until now. We set out to characterize these subunits individually or in combination by using a cDNA expression system in cultured mammalian cells. Coexpression of p68 markedly increased the protein level of p180, with the result that ectopically generated DNA polymerase activity was dramatically increased. Immunofluorescence analysis showed that while either singly expressed p180 or p68 was localized in the cytoplasm, cotransfection of both subunits resulted in colocalization in the nucleus. We identified a putative nuclear localization signal for p180 (residues 1419 to 1437) and found that interaction with p68 is essential for p180 to translocate into the nucleus. These results indicate that association of p180 with p68 is important for both protein synthesis of p180 and translocation into the nucleus, implying that p68 plays a pivotal role in the newly synthesized DNA polymerase alpha complex.

3T3 Cells↗
PubMed1998

Magnetic resonance imaging of the shoulder in abduction.

To simulate the painful arm position in subacromial impingement syndrome, magnetic resonance imaging of the shoulder was done with the arms at the sides and at 80 degrees abduction in two groups of patients. Group A had 20 shoulders in 20 patients with subacromial impingement syndrome and Group B had 19 shoulders in 19 patients without impingement syndrome. When arm position was changed from adduction to abduction, an increase in signal intensity of the rotator cuff tendon was seen more often in Group A (45%) than in Group B (26%). Group A shoulders showed encroachment of the acromion or the acromioclavicular joint to the rotator cuff in 25% of the shoulders with the arms at the sides, and in 75% with the arms in abduction. Similar findings were observed in only 14% and 21% of Group B shoulders in the respective arm positions. Abduction of the arms seemed to cause encroachment of the overlying structure, especially of the acromioclavicular joint, to the rotator cuff in patients with subacromial impingement syndrome, even if there were no such findings in the images with the arms at the sides.

Acromioclavicular Joint↗
PubMed1998

Septic arthritis of a lumbar facet joint: report of a case with early MRI findings.

A 56-year-old man was hospitalized with a 2-day history of back pain and fever. A magnetic resonance (MR) scan taken on admission showed a facet joint effusion and soft-tissue abnormality posterior to the joint. Follow-up MR imaging confirmed the facet joint destruction and periarticular abscess. A biopsy specimen grew Staphylococcus aureus. MR imaging is helpful in detecting this rare infection in the earliest phase of the disease.

Abscess↗
PubMed1997

Overexpression of 3'-untranslated region of the myotonic dystrophy kinase cDNA inhibits myoblast differentiation in vitro.

The genetic defect underlying myotonic dystrophy (DM) has been identified as an unstable CTG trinucleotide repeat amplification in the 3'-untranslated region (3'-UTR) of the DM kinase gene (DMK). Individuals with the most severe congenital form display a marked delay in muscle terminal differentiation. To gain insight into the role of DMK during myogenesis, we have examined the effect of DMK overexpression on the terminal differentiation of the murine myoblast cell line C2C12. We demonstrate that a 4-10-fold constitutive overexpression of DMK mRNA in myoblasts caused a marked inhibition of terminal differentiation. Surprisingly, this activity was mapped to a 239-nucleotide region of the 3'-UTR of the DMK transcript. When the DMK 3'-UTR was placed downstream of a reporter gene, the same inhibition of myogenesis was observed. Following the induction of differentiation of myoblast clones overexpressing the DMK 3'-UTR, the levels of myogenin mRNA were reduced by approximately 4-fold, whereas the steady state levels of mef-2c transcripts were not affected. These data suggest that overexpression of the DMK 3'-UTR may interfere with the expression of musclespecific mRNAs leading to a delay in terminal differentiation.

3T3 Cells↗
PubMed1997

Distribution of neuronal apoptosis inhibitory protein-like immunoreactivity in the rat central nervous system.

We have recently shown that spinal muscular atrophy (SMA), an autosomal recessive disorder characterized by motor neuron loss, is associated with deletion of a gene that encodes the neuronal apoptosis inhibitory protein (NAIP). In the present study, we have examined the distribution of NAIP-like immunoreactivity (NAIP-LI) in the rat central nervous system (CNS) by using an affinity-purified polyclonal antibody against NAIP. In the forebrain, immunoreactive neurons were detected in the cortex, the hippocampus (pyramidal cells, dentate granule cells, and interneurons), the striatum (cholinergic interneurons), the basal forebrain (ventral pallidum, medial septal nucleus, and diagonal band), the thalamus (lateral and ventral nuclei), the habenula, the globus pallidus, and the entopenduncular nucleus. In the midbrain, NAIP-LI was located primarily within neurons of the red nucleus, the substantia nigra pars compacta, the oculomotor nucleus, and the trochlear nucleus. In the brainstem, neurons containing NAIP-LI were observed in cranial nerve nuclei (trigeminal, facial, vestibular, cochlear, vagus, and hypoglossal nerves) and in relay nuclei (pontine, olivary, lateral reticular, cuneate, gracile nucleus, and locus coeruleus). In the cerebellum, NAIP-LI was found within both Purkinje and nuclear cells (interposed and lateral nuclei). Finally, within the spinal cord, NAIP-LI was detected in Clarke's column and in motor neurons. Taken together, these results indicate that NAIP-LI is distributed broadly in the CNS. However, high levels of NAIP-LI were restricted to those neuronal populations that have been reported to degenerate in SMA. This anatomical correspondence provides additional evidence for NAIP involvement in the neurodegeneration observed in acute SMA.

Animals↗
PubMed1997

DJ-1, a novel oncogene which transforms mouse NIH3T3 cells in cooperation with ras.

We have isolated and characterized the cDNA encoding a novel protein designated DJ-1. DJ-1, sharing no significant homology with the sequences so far reported, did not show transactivation activity in the Gal4 recombinant system, but transformed mouse NIH3T3 cells by itself. Furthermore, DJ-1 showed a cooperative transforming activity with H-Ras, more than 3 times as strong as the activity of ras/myc combination. DJ-1 was ubiquitously expressed in various human tissues, and the expression was induced by growth stimuli. Moreover, DJ-1 translocated from cytoplasm to nuclei in the S phase of the cell cycle. DJ-1 is thus suggested to be a novel mitogen-dependent oncogene product involved in a Ras-related signal transduction pathway.

3T3 Cells↗
PubMed1997

Protective effects of local hypothermia in vitrectomy under fluctuating intraocular pressure.

The effects of local hypothermia on the blood-aqueous barrier and retina were investigated in experimental vitrectomy under wide-ranging fluctuations of intraocular pressure. After anesthetized albino rabbits had undergone closed vitrectomy, their vitreous cavities were continuously irrigated for 60 minutes. Perfusion pressures were fluctuated alternately between 10 and 80 mmHg. The rabbits were divided into two groups in accordance with their intraocular perfusion temperatures: 8 degrees C and 22 degrees C. Electroretinograms were taken both before and after the irrigation. Protein and prostaglandin E2 (PGE2) concentrations in the aqueous humor were measured on the 1st and 7th postoperative days. Histologic examination was performed on the eyes enucleated on the same days. There were no significant differences in either a- or b-wave amplitudes between the 8 degrees C and 22 degrees C groups. Both aqueous protein and PGE2 concentrations were significantly higher in the 22 degrees C group than in the 8 degrees C group on the 1st postoperative day. Histologically, the ciliary body showed remarkably more stromal edema in the 22 degrees C group than in the 8 degrees C group. The severity of the retinal damage did not seem to differ between the two groups. Local hypothermia during vitrectomy under fluctuating intraocular pressure inhibits the breakdown of the blood-aqueous barrier in the early postoperative stages.

Animals↗
PubMed1997

A keratin K10 gene mutation in a Japanese patient with epidermolytic hyperkeratosis.

Epidermolytic hyperkeratosis (EHK), or bullous congenital ichthyosiform erythroderma, is characterized by generalized erythroderma, ichthyosiform skin and blistering, and is caused by an aberration of the keratin intermediate filaments. In this study, we examined keratin K10 and 1 gene mutations in a Japanese EHK patient who had severe ichthyosiform erythroderma at birth and developed subsequent blistering. The patient had a G to A transition at codon 156 of the keratin K10 gene, which resulted in an arginine (Arg)-->histidine (His) substitution in the helix initiation peptide of the highly-conserved 1A domain in keratin K10. This is the first mutation report of a Japanese patient with EHK, although the position and mode of the mutation identified here did not differ from those in reported Western cases.

Amino Acid Sequence↗