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Akira Kobayashi

Publications and source records attributed to Akira Kobayashi.

At least 19 recordsLinked to original sources
PubMed2004

Identification of polymorphisms in the promoter region of the human NRF2 gene.

Transcription factor Nrf2 regulates the basal and inducible expression of detoxifying and antioxidant genes. Recent studies using nrf2-null mice suggest that Nrf2 dysfunction might be involved in the pathogenesis of human diseases. To gain insight into the relationship between impairment in the NRF2 gene and human diseases, we attempted to identify polymorphisms in the human NRF2 gene. We determined the structure of the NRF2 gene and found three single nucleotide polymorphisms and one triplet repeat polymorphism in its regulatory region. These results provide a molecular basis for the genetic analysis of the NRF2 gene. The frequency of each polymorphism was examined in two groups of patients with systemic lupus erythematosus and chronic obstructive pulmonary disease. This study did not reveal a close connection between the risk of these diseases and the polymorphisms. However, available lines of evidence suggest the importance of examining the link between NRF2 polymorphisms and other oxidative stress-related diseases.

Base Sequence↗
PubMed2004

The petunia ortholog of Arabidopsis SUPERMAN plays a distinct role in floral organ morphogenesis.

Arabidopsis (Arabidopsis thaliana) SUPERMAN (SUP) plays a role in establishing a boundary between whorls 3 and 4 of flowers and in ovule development. We characterized a Petunia hybrida (petunia) homolog of SUP, designated PhSUP1, to compare with SUP. Genomic DNA of the PhSUP1 partially restored the stamen number and ovule development phenotypes of the Arabidopsis sup mutant. Two P. hybrida lines of transposon (dTph1) insertion mutants of PhSUP1 exhibited increased stamen number at the cost of normal carpel development, and ovule development was defective owing to aberrant growth of the integument. Unlike Arabidopsis sup mutants, phsup1 mutants also showed extra tissues connecting stamens, a petal tube and an ovary, and aberrancies in the development of anther and placenta. PhSUP1 transcripts occurred in the basal region of wild-type flowers around developing organ primordia in whorls 2 and 3 as well as in the funiculus of the ovule, concave regions of the placenta, and interthecal regions of developing anthers. Overexpression of PhSUP1 in P. hybrida resulted in size reduction of petals, leaves, and inflorescence stems. The shortening of inflorescence stems and petal tubes was primarily attributable to suppression of cell elongation, whereas a decrease in cell number was mainly responsible for the size reduction of petal limbs.

Amino Acid Sequence↗
PubMed2004

Changes in sugar content and activity of vacuolar acid invertase during low-temperature storage of potato tubers from six Japanese cultivars.

The storage of potato tubers at low temperatures leads to the accumulation of sugars in a process called "low-temperature sweetening." To understand this phenomenon, we measured the sugar contents and the activity of acid invertase over several months in tubers of six Japanese cultivars stored at 4 degrees C or 20 degrees C. At 20 degrees C, few changes in sugar contents took place in any of the tubers. On the other hand, when stored at 4 degrees C, three types of changes were observed among the cultivars: (1) increased levels of reducing sugars during storage; (2) a pattern similar to that of type 1, but with 4- to 6-fold lower levels of reducing sugars throughout storage; and (3) increased sucrose, but not reducing sugars. The activity of vacuolar acid invertase increased in the type-1 cultivars, whereas, in the type-2 and type-3 cultivars, the activities were very low during storage at 4 degrees C. Reverse transcription-polymerase chain reaction analysis of acid invertase showed that the transcripts of the enzyme accumulated in the tubers stored at 4 degrees C in the type-1 cultivars but not in type-3. These results suggest that the activity of vacuolar acid invertase is related to the types of changes that occurred in sugar content during low-temperature storage among the potato cultivars.

Cold Temperature↗
PubMed2004

Protection against electrophile and oxidant stress by induction of the phase 2 response: fate of cysteines of the Keap1 sensor modified by inducers.

Induction of a family of phase 2 genes encoding for proteins that protect against the damage of electrophiles and reactive oxygen intermediates is potentially a major strategy for reducing the risk of cancer and chronic degenerative diseases. Many phase 2 genes are regulated by upstream antioxidant response elements (ARE) that are targets of the leucine zipper transcription factor Nrf2. Under basal conditions, Nrf2 resides mainly in the cytoplasm bound to its cysteine-rich, Kelch domain-containing partner Keap1, which is itself anchored to the actin cytoskeleton and represses Nrf2 activity. Inducers disrupt the Keap1-Nrf2 complex by modifying two (C273 and C288) of the 25 cysteine residues of Keap1. The critical role of C273 and C288 was established by (i) their high reactivity when purified recombinant Keap1 was treated with dexamethasone mesylate and the dexamethasone-modified tryptic peptides were analyzed by mass spectrometry, and (ii) transfection of keap1 and nrf2 gene-deficient mouse embryonic fibroblasts with constructs expressing cysteine to alanine mutants of Keap1, and measurement of the ability of cotransfected Nrf2 to repress an ARE-luciferase reporter. Reaction of Keap1 with inducers results in formation of intermolecular disulfide bridges, probably between C273 of one Keap1 molecule and C288 of a second. Evidence for formation of such dimers was obtained by 2D PAGE of extracts of cells treated with inducers, and by the demonstration that whereas C273A and C288A mutants of Keap1 alone could not repress Nrf2 activation of the ARE-luciferase reporter, an equal mixture of these mutant constructs restored repressor activity.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Scaffolding of Keap1 to the actin cytoskeleton controls the function of Nrf2 as key regulator of cytoprotective phase 2 genes.

Transcription factor Nrf2 regulates basal and inducible expression of phase 2 proteins that protect animal cells against the toxic effects of electrophiles and oxidants. Under basal conditions, Nrf2 is sequestered in the cytoplasm by Keap1, a multidomain, cysteinerich protein that is bound to the actin cytoskeleton. Keap1 acts both as a repressor of the Nrf2 transactivation and as a sensor of phase 2 inducers. Electrophiles and oxidants disrupt the Keap1-Nrf2 complex, resulting in nuclear accumulation of Nrf2, where it enhances the transcription of phase 2 genes via a common upstream regulatory element, the antioxidant response element. Reporter cotransfection-transactivation analyses with a series of Keap1 deletion mutants revealed that in the absence of the double glycine repeat domain Keap1 does not bind to Nrf2. In addition, deletion of either the intervening region or the C-terminal region also abolished the ability of Keap1 to sequester Nrf2, indicating that all of these domains contribute to the repressor activity of Keap1. Immunocytochemical and immunoprecipitation analyses demonstrated that Keap1 associates with actin filaments in the cytoplasm through its double glycine repeat domain. Importantly, disruption of the actin cytoskeleton promotes nuclear entry of an Nrf2 reporter protein. The actin cytoskeleton therefore provides scaffolding that is essential for the function of Keap1, which is the sensor for oxidative and electrophilic stress.

Actin Cytoskeleton↗
PubMed2004

Oxygen permeability of amniotic membrane and actual tear oxygen tension beneath amniotic membrane patch.

PURPOSE: To investigate the oxygen permeability (Dk) and oxygen transmissibility (Dk/t) of amniotic membrane (AM), and tear oxygen tension beneath AM in rabbits. DESIGN: Experimental study. METHOD: The water content of AM was measured to calculate Dk and Dk/t, and compared with those of a therapeutic soft contact lens (TSCL). The tear oxygen tension beneath AM and TSCL was also measured in four male albino rabbits. RESULTS: The average water content of AM was significantly higher than that of TSCL (96.8 +/- 0.8% (mean +/- SD) vs 78%, P =.0006), giving rise to a significantly higher Dk and Dk/t in AM as compared with TSCL (142.8 +/- 4.7 vs 65.3, respectively, P =.0012, and 92.9 +/- 3.0 vs 29.7, respectively, P =.0008). The average tear oxygen tension under AM was also higher than TSCL (94.9 +/- 2.9 vs 59.1 +/- 4.9 mm Hg, P =.0006). CONCLUSIONS: These findings support the rationale of using AM as a superior bandage in treating persistent corneal epithelial defects or ulcers.

Amnion↗
PubMed2004

Dendritic cells, T-cell infiltration, and Grp94 expression in cholangiocellular carcinoma.

Although dendritic cells (DCs) play an important role in tumor immunity, there have been no reports on their role in cholangiocellular carcinoma (CCC). In 26 formalin-fixed, paraffin-embedded tissue sections from patients with CCC, cells positive for CD83 (a marker of mature DCs), CD1a (a marker of immature DCs), and CD8 and CD4 (T cell markers) were counted, and expression of glucose-regulated protein (grp) 94, which is considered to participate in the maturation of DCs, was evaluated by immunohistochemistry and Western blot analysis to study the relationship between their expression and patients' disease outcome. The number of CD83-positive DCs at the invasive margin of CCCs correlated significantly with the number of CD8-positive or CD4-positive T cells in the cancerous region and was significantly higher in grp94-positive cancer than in grp94-negative cancer (P = 0.0006). CD83-positive patients (positive cells in invasive margin > 12.4/field) had both a significantly lower incidence of lymph node metastasis (23.1% vs 69.2%; P = 0.0206) and a better outcome than CD83-negative patients (P <0.001). We conclude that mature DCs are distributed predominantly at the invasive margin of cancers, and a significantly higher number of mature DCs at the invasive margin are observed in patients with grp94-positive cancer cells. Mature DCs may enhance CD8- and CD4-positive cell infiltration into cancers and improve prognosis in patients with CCC, due in part to abatement of lymph node metastasis.

Aged↗
PubMed2004

Reliability of intraocular pressure by Tono-Pen XL over amniotic membrane patch in human.

PURPOSE: To determine the reliability of Tono-Pen XL in measuring intraocular pressure (IOP) over the amniotic membrane patch in human eyes. METHOD: Ten healthy volunteers (nine males and one female) participated in this study. After topical anesthesia, IOP was measured using the Tono-Pen XL before and after the application of a single-layer amniotic membrane patch in the right eye and double-layer amniotic membrane patch in the left eye. RESULTS: We found no statistical difference between the IOP measured with and without a single layer amniotic membrane (P = 0.808). Nor did we find any statistical difference between the IOP measured with and without double-layer amniotic membrane patch (P = 0.813). The average thickness of the single- and the double-layer amniotic membrane patch measured by pachymetry was 131.9 +/- 31.4 microm (mean +/- SD) and was 246.1 +/- 69.3 microm (mean +/- SD), respectively. CONCLUSIONS: This study showed that it is reliable to measure IOP by Tono-Pen XL over single or a double layer of amniotic membrane patch on healthy human eyes. Further studies are required to determine the accuracy of IOP measurement over amniotic membrane patch on diseased corneas.

Adult↗
PubMed2004

Marked and sustained increase in bone mineral density after parathyroidectomy in patients with primary hyperparathyroidism; a six-year longitudinal study with or without parathyroidectomy in a Japanese population.

OBJECTIVE: Although many reports have demonstrated the sustained increase in bone mineral density (BMD) at trabecular sites in primary hyperparathyroidism (pHPT) after parathyroidectomy (PTX), there have been no data available on BMD changes over the long-term in pHPT patients with and without PTX in Japanese population. The present study was designed to investigate long-term BMD changes at both trabecular and cortical sites in Japanese pHPT patients with or without PTX. METHODS: The subjects were 97 patients who had been followed up in Kobe University Hospital for at least 1 year up to 6 years with or without PTX. PTX was recommended to all patients whose pathological parathyroid gland(s) could be determined by image diagnosis. BMD was measured at the lumbar spine (L2-L4) and at distal one-third of the radius (R1/3) by dual energy X-ray absorptiometry (QDR2000). Serum levels of calcium, alkaline phosphatase and parathyroid hormone (PTH) were determined at the time of the BMD measurement. RESULTS: Significant increases in any of the indices of BMD from the baseline values were observed within three months after PTX, followed by sustained increases over 6 years at L2-L4 even in postmenopausal women. Radial BMD also showed a marked increase six years after PTX. L2-L4 eventually reached the normal BMD but R1/3 did not. The percentage changes in L2-L4 were positively and significantly correlated with the preoperative PTH levels over the study period. However, the percentage changes in R1/3 showed a significant correlation with the preoperative PTH levels only 5 and 6 years after PTX. In the patients without PTX, no obvious changes in biochemical indices and BMD were observed over the six years. CONCLUSION: We demonstrated that PTX led to marked and sustained increases in BMD not only at L2-L4 but also at R1/3 in Japanese pHPT patients, including postmenopausal women. The preoperative PTH level could be a clinically useful index for predicting long-term BMD changes after PTX.

Bone Density↗
PubMed2004

Oxidative stress sensor Keap1 functions as an adaptor for Cul3-based E3 ligase to regulate proteasomal degradation of Nrf2.

Transcription factor Nrf2 is a major regulator of genes encoding phase 2 detoxifying enzymes and antioxidant stress proteins in response to electrophilic agents and oxidative stress. In the absence of such stimuli, Nrf2 is inactive owing to its cytoplasmic retention by Keap1 and rapid degradation through the proteasome system. We examined the contribution of Keap1 to the rapid turnover of Nrf2 (half-life of less than 20 min) and found that a direct association between Keap1 and Nrf2 is required for Nrf2 degradation. In a series of domain function analyses of Keap1, we found that both the BTB and intervening-region (IVR) domains are crucial for Nrf2 degradation, implying that these two domains act to recruit ubiquitin-proteasome factors. Indeed, Cullin 3 (Cul3), a subunit of the E3 ligase complex, was found to interact specifically with Keap1 in vivo. Keap1 associates with the N-terminal region of Cul3 through the IVR domain and promotes the ubiquitination of Nrf2 in cooperation with the Cul3-Roc1 complex. These results thus provide solid evidence that Keap1 functions as an adaptor of Cul3-based E3 ligase. To our knowledge, Nrf2 and Keap1 are the first reported mammalian substrate and adaptor, respectively, of the Cul3-based E3 ligase system.

Animals↗
PubMed2003

Photoreceptor synaptic protein HRG4 (UNC119) interacts with ARL2 via a putative conserved domain.

Human retinal gene 4 (HRG4) (UNC119) is a photoreceptor synaptic protein of unknown function, shown when mutated to cause retinal degeneration in a patient and in a confirmatory transgenic model. ADP-ribosylation factor-like protein 2 (ARL2) was identified as an interactor of HRG4 by the yeast two-hybrid strategy. The presence of ARL2 in the retina and co-localization with HRG4 was confirmed by Western blot and double immunofluorescence analysis, respectively. The interaction of ARL2 with HRG4 was further confirmed by co-immunoprecipitation and direct binding analysis. Phosphodiesterase delta (PDEdelta) is an ARL2-binding protein homologous to HRG4. Amino acid residues of PDEdelta involved in binding ARL2 and forming a hydrophobic pocket were shown to be highly conserved in HRG4, suggesting similarity in binding mechanism and function.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2003

Accuracy of intraocular pressure by Tono-Pen XL over amniotic membrane patching in rabbits.

PURPOSE: To report an accuracy of intraocular pressure (IOP) by Tono-Pen XL over amniotic membrane patching in rabbits. DESIGN: Experimental study. METHODS: Seven male albino rabbits were used in this study. After general anesthesia, IOP was measured in both eyes using the Tono-Pen without amniotic membrane. A single layer amniotic membrane was then immediately placed over the rabbit eye, and the IOP measurement was repeated. RESULTS: The average IOP without amniotic membrane was 5.8 +/- 0.6 mm Hg (mean +/- SD). The average IOP through amniotic membrane was 6.0 +/- 0.4 mm Hg (mean +/- SD). There was no statistical difference in IOP measured on corneas with or without the amniotic membrane (P =.34). CONCLUSIONS: The accuracy of IOP measurement by Tono-Pen XL over a single layer of amniotic membrane patching was demonstrated using rabbit eyes. Further studies in human eyes may verify our findings in actual clinical settings.

Amnion↗
PubMed2003

Detection of rabbit corneal epithelium over amniotic membrane patch by white-light and laser confocal microscopy.

PURPOSE: To report confocal microscopic observation of rabbit corneal epithelium through amniotic membrane patch (AMP). DESIGN: Experimental study. METHOD: Six rabbit eyes were patched by amniotic membrane. Then, white-light and laser confocal microscopic observation was performed. RESULTS: Images of human amniotic epithelium, amniotic basement membrane, and amniotic stroma were obtained using both devices, followed by detection of rabbit corneal epithelium. CONCLUSION: This study indicates that observation of corneal epithelium through AMP is possible in rabbit eyes using both these methods. This implies that both devices might be used clinically to observe the epithelial healing process under an AMP to better determine when it should be removed.

Amnion↗
PubMed2003

Endoillumination-assisted cataract surgery in a patient with corneal opacity.

We present a technique to better visualize cataracts through corneal opacity. A 70-year-old Japanese woman with retinal detachment, cataract, and corneal opacity in the left eye was treated with phacoemulsification, 3-port vitrectomy, and intraocular lens implantation. To circumvent the difficulty of performing continuous curvilinear capsulorhexis (CCC) and subsequent cataract surgery through a hazy cornea, we used an endoilluminator as a light source outside the cornea for CCC and inside the anterior chamber for phacoemulsification. As a result, CCC and subsequent cataract surgery were successfully and easily performed despite potentially troublesome corneal opacity. The use of an endoilluminator can be an effective supportive measure for cataract surgery in patients with corneal opacity.

Aged↗
PubMed2003

In vivo confocal microscopy in the patients with cornea farinata.

PURPOSE: To report in vivo corneal confocal microscopic findings of patients with cornea farinata. PATIENTS AND METHODS: Two unrelated patients, a 47-year-old man and a 77-year-old woman, with cornea farinata were studied. Examination with a confocal microscope was performed in addition to routine slit-lamp biomicroscopy. RESULTS: In both cases, slit-lamp biomicroscopy showed numerous small, faint opacities in the deep stroma in both eyes. Using confocal microscopy, highly reflective small particles were observed in the cytoplasm of keratocytes in the deep stroma adjacent to the corneal endothelial layer. No abnormalities could be detected in the epithelial layer, in the mid-stromal layer, at the level of Descemet's membrane, and in the endothelial layer. CONCLUSIONS: In vivo corneal confocal microscopy is useful for observing stromal abnormalities in cornea farinata. Further investigation of posterior stromal opacities using confocal microscopy may be useful to understand and differentiate various corneal conditions involving primarily deep stromal layers.

Aged↗