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Takashi Kojima

Publications and source records attributed to Takashi Kojima.

At least 37 records · Page 2Linked to original sources
PubMed2006

[Analysis of the chemical drugs among structural isomer].

Drugs that have a pharmacological effect similar to legal drugs such as narcotics and stimulants are available in the market and widely used. 5-methoxy-N,N-di-iso-propyl-tryptamine (5MeO-DIPT) and alpha-methyl-tryptamine (AMT) were categorized as narcotics and were specified as legal drugs in April 2005, and also 2,5-dimethoxy-4-n-propylthiophenethylamine (4C-T-7) and N-methyl-alpha-ethyl-3,4-methylenedioxy-phenethylamine (MBDB) were categorized as narcotics and were specified as legal drugs in April 2006, in Japan. We are analyzing these chemical drugs by investigating the market research. It is recognized that during the analysis of chemical drugs, drugs that resemble a structural isomer of a target substance, such as 5MeO-DIPT and 5-methoxy-N,N-di-n-propyl-tryptamine (5MeO-DPT) or 4C-T-7 and 2,5-dimethoxy-4-iso-propylthiophenethylamine (4C-T-4), should be distinguished. The results of TLC, IR, GC-MS and HPLC analyses were compared. 5MeO-DIPT and 5MeO-DPT could be distinguished by TLC and HPLC analyses, but not by IR and GC-MS analysis. The drugs 4-hydroxy-N-methyl-N-iso-propyl-tryptamine (4HO-MIPT) and 4-hydroxy-N,N-di-ethyl-tryptamine (4HO-DET) or could not be distinguished. Moreover, the isomers of 4-hydroxy-N-methyl-N-n-propyl-tryptamine (4HO-MPT) was not found to be present. Thus, we have demonstrated that the chemical drug could be distinguished from each other, and we have also shown that NMR data is essential for the analysis.

Chromatography, High Pressure Liquid↗
PubMed2005

Structural transition of glucagon in the concentrated solution observed by electrophoretic and spectroscopic techniques.

Glucagon, a polypeptide hormone consisting of 29 amino acid residues, tends to form gel-like fibrillar aggregates, and the glucagon fibril, as well as other pathologically related fibrils including prion, amylin, and beta-amyloid, have been found to be cytotoxic through the activation of apoptotic signaling pathways. To understand the aggregation properties of glucagon fibril, we have characterized and compared the physicochemical properties of glucagon, secretin, a member of the glucagon superfamily, and amylin using analytical techniques including capillary electrophoresis (CE), circular dichroism (CD), FT-IR, FT-Raman, transmission electron microscopy (TEM), and beta-sheet-imaging probe. Aging treatment of glucagon resulted in the formation of fibrillar aggregates in time- and concentration-dependent manner, and FT-IR and FT-Raman analyses showed the spectral shift of amide I band, suggesting the conformational changes from alpha-helix to beta-sheet structure. Interestingly, secretin, having high sequential and secondary structural homology with glucagon, did not generate the fibrillar aggregates at the conditions tested. In addition, we evaluated the association state of glucagon at various pHs raging from pH 2.0 to 3.5 using CE. Based on the CE data, the rate constants of glucagon aggregation were calculated to be 0.002 +/- 0.004/h and 0.080 +/- 0.011/h for aging at pH 2.0 and 3.5, respectively, suggesting the pH dependence of self-association. CE showed the potential to separate and detect the glucagon aggregates and intermediates during aging process.

Amino Acid Sequence↗
PubMed2005

Phosphorylation of ezrin enhances microvillus length via a p38 MAP-kinase pathway in an immortalized mouse hepatic cell line.

The apical microvilli are closely related with the development and the maintenance of cell polarization, and the length of microvilli varies in a regular way among cell types. Ezrin, a member of the ezrin/radixin/moesin (ERM) family, seems to be involved in the formation and stabilization of the apical microvilli. We found that phosphorylation of ezrin caused elongation of microvilli via a p38 MAP-kinase signaling pathway in an immortalized mouse hepatic cell line. When, in the oncogenic Raf-1-transfected mouse hepatic cell line, epithelial to mesenchymal transition (EMT) indicated as down-regulation of E-cadherin and up-regulation of Snail occurred, loss of microvilli and down-regulation of ezrin but not radixin and moesin were also observed. In the Raf-1 transfectants treated with the MAP-kinase inhibitor PD98059 and the p38 MAP-kinase inhibitor SB203580, the numbers of microvilli and the expression of ezrin, E-cadherin and Snail were recovered. More interestingly, treatment with SB203580 induced elongation of microvilli and increased phosphorylation of ezrin (at Thr-567 and Tyr-353). Phosphorylated ezrin-positive dots were colocalized with actin-positive dots on the surface of some Raf-1 transfectants treated with SB203580. These results suggested that phosphorylation of ezrin via the p38 MAP-kinase signaling pathway might be involved in the formation of microvilli during development of epithelial cell polarization.

Acrylates↗
PubMed2005

Behavior of tight-junction, adherens-junction and cell polarity proteins during HNF-4alpha-induced epithelial polarization.

We previously reported that expression of tight-junction molecules occludin, claudin-6 and claudin-7, as well as establishment of epithelial polarity, was triggered in mouse F9 cells expressing hepatocyte nuclear factor (HNF)-4alpha [H. Chiba, T. Gotoh, T. Kojima, S. Satohisa, K. Kikuchi, M. Osanai, N. Sawada. Hepatocyte nuclear factor (HNF)-4alpha triggers formation of functional tight junctions and establishment of polarized epithelial morphology in F9 embryonal carcinoma cells, Exp. Cell Res. 286 (2003) 288-297]. Using these cells, we examined in the present study behavior of tight-junction, adherens-junction and cell polarity proteins and elucidated the molecular mechanism behind HNF-4alpha-initiated junction formation and epithelial polarization. We herein show that not only ZO-1 and ZO-2, but also ZO-3, junctional adhesion molecule (JAM)-B, JAM-C and cell polarity proteins PAR-3, PAR-6 and atypical protein kinase C (aPKC) accumulate at primordial adherens junctions in undifferentiated F9 cells. In contrast, CRB3, Pals1 and PATJ appeared to exhibit distinct subcellular localization in immature cells. Induced expression of HNF-4alpha led to translocation of these tight-junction and cell polarity proteins to beltlike tight junctions, where occludin, claudin-6 and claudin-7 were assembled, in differentiated cells. Interestingly, PAR-6, aPKC, CRB3 and Pals1, but not PAR-3 or PATJ, were also concentrated on the apical membranes in differentiated cells. These findings indicate that HNF-4alpha provokes not only expression of tight-junction adhesion molecules, but also modulation of subcellular distribution of junction and cell polarity proteins, resulting in junction formation and epithelial polarization.

Adherens Junctions↗
PubMed2005

Down-regulation of survival signaling through MAPK and Akt in occludin-deficient mouse hepatocytes in vitro.

The tight junction (TJ) regulates epithelial cell polarity and barrier including permeability of the paracellular pathway. Occludin was the first integral membrane protein to be discovered, but it is not indispensable for the formation of TJ strands. The physiological function of occludin is still unclear, although occludin-deficient mice show very complex abnormalities in various organs without overt dysfunction of the TJ. To investigate the role of occludin in TJ expression and apoptosis regulated by survival signal transduction pathways such as MAPK and Akt, we performed primary culture of hepatocytes and established hepatic cell lines from occludin-deficient mice. In primary cultures of occludin-deficient mouse hepatocytes, claudin-2 expression and apoptosis were induced by down-regulation of the activation of MAPK and Akt. In the hepatic cell lines derived from occludin-deficient mice, claudin-2 expression and serum-free induced apoptosis were also increased by down-regulation of the activation of MAPK and Akt. Furthermore, in the hepatic cell lines transiently transfected with mouse and rat occludin genes, induction of claudin-2 expression and the apoptosis were inhibited with increases in activation of MAPK and Akt. These findings show that occludin plays a crucial role in claudin-2-dependent TJ function and the apoptosis involving MAPK and Akt signaling pathways in hepatocytes.

Animals↗
PubMed2005

Glyceraldehyde-derived advanced glycation end-products preferentially induce VEGF expression and reduce GDNF expression in human astrocytes.

The blood-brain barrier (BBB) is a biological unit composed of capillary endothelial cells and astrocytes. Here we examined the effects of various types of advanced glycation end-products (AGEs) on astrocytes and BBB-forming endothelial cells. While no type of AGE we examined changed the permeability of endothelial sheets, glyceraldehyde-derived AGE induced VEGF expression most significantly in astrocytes. The expression of glial cell line-derived neurotrophic factor (GDNF), which reduces the vascular permeability, was decreased in the astrocytes by treatment with glyceraldehyde-derived AGE. These results indicate that glyceraldehyde-derived AGE is the biologically active substance for astrocytes by regulating the VEGF and GDNF expression, which is causally contributing to an increase in the permeability of the BBB.

Astrocytes↗
PubMed2005

Activation of p21CIP1/WAF1 gene expression and inhibition of cell proliferation by overexpression of hepatocyte nuclear factor-4alpha.

The F9 murine embryonal carcinoma cell line provides an attractive system for studying epithelial differentiation and antiproliferative processes. We have recently established F9 cells expressing doxycycline-inducible hepatocyte nuclear factor (HNF)-4alpha and shown that HNF-4alpha triggers the gene expression of tight-junction molecules, occludin, claudin-6, and claudin-7, as well as formation of functional tight junctions and polarized epithelial morphology (Exp. Cell Res. 286, [2003] 288). Since these events were very similar to those induced by retinoids, we investigated whether HNF-4alpha, like retinoid receptors, was involved in the control of cell proliferation. We herein show that HNF-4alpha up-regulates expression of the p21 gene, but not the p15, p16, p18, p19, or p27 gene, in a p53-independent manner, and inhibits cell growth in F9 cells. Similar results were observed in rat lung endothelial cells, in which expression of HNF-4alpha is conditionally induced by doxycycline. Furthermore, we demonstrate, by reporter assay, that HNF-4alpha significantly elevates the transcriptional activity of the p21 promoter. Since, HNF-4alpha is expressed not only in the liver but also in organs containing epithelial cells, such as kidney, intestine, pancreas, and stomach, it might also play critical roles in the regulation of epithelial morphogenesis and proliferation in these organs.

Animals↗
PubMed2005

The effect of autologous serum eyedrops in the treatment of severe dry eye disease: a prospective randomized case-control study.

PURPOSE: To evaluate the effectiveness of the autologous serum eyedrops in the treatment of severe dry eye patients. DESIGN: Prospective randomized case-control study. METHODS: Thirty-seven eyes of twenty severe dry eye patients without punctal occlusion were enrolled in this study. After 2 weeks of washout, they were randomly assigned to two groups. Group A patients used only preservative-free artificial tears, and group S patients used only autologous serum eyedrops. We evaluated the results of Schirmer test, fluorescein and rose bengal staining scores, tear film breakup time (BUT), and subjective symptom scores before and 2 weeks after treatment. RESULTS: Mean BUT and fluorescein and rose bengal staining scores, as well as subjective symptom scores, showed significant improvement in the patients assigned to autologous serum eyedrops compared with subjects assigned to preservative-free artificial tears after 2 weeks of treatment. CONCLUSIONS: Autologous serum eyedrops were found effective in the treatment of severe dry eye disease, as evidenced by improvement of tear stability and ocular surface vital staining scores.

Aged↗
PubMed2005

The application of a new continuous functional visual acuity measurement system in dry eye syndromes.

PURPOSE: To evaluate the efficacy of a new continuous functional visual acuity measurement (FVAM) system for the assessment of dry eye patients. DESIGN: Prospective comparative study. METHODS: Monocular recognition acuity measured continuously by the FVAM system during a 30-second blink-free period was defined as functional visual acuity (FVA). Examinations using the FVAM system were conducted in 35 eyes of 20 healthy controls and 19 eyes of 13 dry eye patients. Tear function examinations including the Schirmer test, tear film break-up time, and fluorescein and Rose Bengal staining were performed in all subjects. Functional visual acuity and tear functions were also examined before and after insertion of punctum plugs in dry eye patients. Functional visual acuity results at 10, 20, and 30 seconds were compared. RESULTS: Functional visual acuity in dry eyes were significantly lower than control subjects at all time points (P < .05). Functional visual acuity after punctum plugs insertion improved significantly at all time points (P < .05). CONCLUSIONS: FVAM system seemed not only to be an effective tool in the assessment of dynamic visual acuity changes in dry eye and normal subjects but in evaluating the outcome of management of dry eye disease by punctum plugs.

Adult↗
PubMed2005

p38 MAP-kinase regulates function of gap and tight junctions during regeneration of rat hepatocytes.

BACKGROUND/AIMS: Hepatocyte regeneration is considered to be associated with adaptive changes in expression of gap and tight junctions through multiple signal transduction pathways including p38 MAP-kinase. The role of the stress responsitive MAP-kinase, p38 MAP-kinase, signaling pathway in function of gap and tight junctions was examined during regeneration of rat hepatocytes in vivo and in vitro. METHODS: We examined changes in formation, expression and function of gap and tight junctions in rat livers after 70% partial hepatectomy and in primary cultures of rat hepatocytes, by using a p38 MAP-kinase inhibitor, SB203580. RESULTS: When p38 MAP-kinase was activated during partial hepatectomy, down-regulation of Cx32 and up-regulation of claudin-1 were observed. By SB203580 treatment, the down-regulation of Cx32 was inhibited and the up-regulation of claudin-1 was enhanced, well maintaining the structures of gap and tight junctions. SB203580 treatment did not affect the increase of hepatocyte proliferation. In EGF induced proliferative rat hepatocytes treated with SB203580, the expression and function of Cx32 and claudin-1 were increased. CONCLUSIONS: Dynamic changes of formation of gap and tight junctions during regeneration of rat hepatocytes in vivo and in vitro are in part controlled via a p38 MAP-kinase signaling pathway, and are independent of cell growth.

Animals↗
PubMed2005

Activity of gelatins to induce secretion of a variety of cytokines from murine peritoneal exudate macrophages.

Previously, we observed that liquid form bovine bone (BB) gelatin stimulates murine spleen cells to proliferate in vitro. In this study, activity of BB gelatin to stimulate murine-adherent peritoneal exudate cells (PEC) to secrete cytokines has been examined. Quantitatively, BB gelatin stimulated adherent PEC of C3H/HeN mice to secrete interleukin (IL)-12 (+p40), TNF-alpha, and IL-6 but not IL-1beta, IL-2, IL-10, and IFN-gamma. Qualitatively, BB gelatin-induced secretion of KC, MIP-2, MCP-1, RANTES, and MIP-1a as well as IL-6 but not 6Ckine, CTACK, Eotaxin, G-CSF, GM-CSF, IL-2,-3,-4,-5,-9,-10,-12,-13,-17, Leptin, IFN-gamma, SCF, sTNFri, TARC, TNF-alpha, TIMP-1, Tpo, and VEGF. BB gelatin acted on adherent PEC of C3H/HeN mice but not C3H/HeJ mice, which lack Toll-like receptor 4. Polymyxin B, a LPS antagonist, did not inhibit the activity of BB gelatin. Lipopolysaccharide (LPS) but not BB gelatin induced secretion of an appreciable amount of mIL-1beta. These results suggest that the activity of BB gelatin is not attributed to contamination of LPS but BB gelatin itself. It was also suggested that BB gelatin stimulated adherent PEC to newly produce and secrete cytokines.

Animals↗
PubMed2005

Tear film and ocular surface abnormalities after eyelid tattooing.

A 45-year-old woman who underwent eyelid tattooing 20 years earlier presented with decreased left vision and eye discomfort. Her ocular history revealed an uneventful LASIK procedure 4 years previously with epithelial problems of the flap followed by S. aureus keratitis. Resultant corneal opacity necessitated a deep lamellar keratoplasty performed 9 months prior to admission followed by frequent epithelial problems. At the time of admission, her left eye had a corneal epithelial defect and both lower eyelid margins revealed subepidermal pigmentation and keratinization. Her initial examination revealed tear instability with increased ocular surface staining scores and advanced tear film lipid layer abnormality in both eyes. Meibography showed bilateral total meibomian gland dropout. Treatment with autologous serum eye drops resulted in full epithelialization. Meibomian gland disease-specific therapy did not result in any change in breakup time, vital staining scores, tear film lipid layer interferometry grades, or glandular dropout state.

Eyelid Diseases↗
PubMed2005

Clinical evaluation of the arched blade for cataract surgery.

PURPOSE: To evaluate the efficacy of the arched blade for making clear corneal incisions in cataract surgery. METHODS: This prospective study comprised 112 eyes of 74 patients scheduled for cataract surgery. Temporal clear corneal incisions were made with either a 3.2-mm conventional flat blade or the arched blade. The choice of knife was randomly assigned. Two surgeons, one with substantial cataract surgery experience and the other with less experience, performed the surgery. Corneal topography and aberration were examined pre- and postoperatively. The degree of surgically induced astigmatism (SIA) and high order aberration was analysed. The self-sealing ability of the wound was also compared between both blades. RESULTS: For the less experienced surgeon, the degree of SIA was significantly higher with the 3.2-mm flat blade than with the 3.2-mm arched blade as measured at any time during postoperative follow-up. For the more experienced surgeon, the degree of high order aberration increased significantly with the 3.2-mm flat blade. The incision's self-sealing ability was significantly better when the wound was made with the arched blade rather than with the flat blade. CONCLUSION: The arched blade proved to be effective in reducing surgically induced astigmatism and high order aberration in cataract surgery, particularly when used by the less experienced surgeon. Using the arched blade should lead to better wound self-sealing and, therefore, safer surgical results.

Aged↗
PubMed2005

HLA-DR- and CD11c-positive dendritic cells penetrate beyond well-developed epithelial tight junctions in human nasal mucosa of allergic rhinitis.

The epithelial barrier of the upper respiratory tract plays a crucial role in host defense. In this study, to elucidate whether there is antigen monitoring by dendritic cells (DCs) beyond the epithelial tight-junction barrier in allergic rhinitis, we investigated the expression and function of tight junctions and characterized DCs in the epithelium of nasal mucosa from patients with allergic rhinitis. In reverse transcription-polymerase chain reaction, mRNAs of tight-junction proteins occludin, JAM-1, ZO-1, and claudin-1, -4, -7, -8, -12, -13, and -14 were detected in the nasal mucosa. Occludin, JAM-1, and ZO-1 were colocalized in the uppermost layer in the pseudostratified epithelium of the nasal mucosa, whereas claudin-1, -4, and -7 were found throughout the epithelium. In freeze-fracture replicas of the nasal mucosa, continuous tight-junction strands formed well-developed networks. Epithelial barrier function measured by a dye tracer was well maintained in occludin-positive tight junctions in the epithelium of the nasal mucosa. HLA-DR- and CD11c-positive DCs expressed claudin-1 and penetrated beyond occludin in the epithelium of the nasal mucosa with, but not without, allergic rhinitis. These results indicate that DCs may easily access antigens beyond epithelial tight junctions in the human nasal mucosa of allergic rhinitis.

Adult↗
PubMed2004

Tight junction protein MAGI-1 is up-regulated by transfection with connexin 32 in an immortalized mouse hepatic cell line: cDNA microarray analysis.

Gap junctions are considered to play a crucial role in differentiation of epithelial cells, including hepatocytes. Recently, we found that Cx32 but not Cx26 was closely related to tight junctional proteins in primary cultured rat hepatocytes (Kojima et al., Exp Cell Res 263:193-201, 2001) and that Cx32 formation and/or Cx32-mediated intercellular communication could induce expression and function of tight junctions in a mouse hepatic cell line (Kojima et al., Exp Cell Res 276:40-51, 2002). In this study, to investigate the mechanisms of induction of tight junctions by transfection with Cx32, we performed cDNA microarray analysis of Cx32 transfectants, compared with parental cells derived from Cx32-deficient hepatocytes. In cDNA microarray analysis, a 2.5-fold increase in expression of membrane-associated guanylate kinase with inverted orientation-1 (MAGI-1), which is known to be localized at adherens and tight junction regions, was observed. High expression of MAGI-1 in Cx32 transfectants was confirmed by Western blotting and RT-PCR. MAGI-1 was colocalized with occludin, claudin-2, ZO-1, and F-actin, but not with E-cadherin in the apical-most regions at cell borders of Cx32 transfectants, similar to junctional adhesion molecule-1 (JAM-1), which may play a crucial role in formation and assembly of tight junctions. Treatment with the gap junction blocker 18beta-glycyrrhetinic acid did not affect expression of MAGI-1 and JAM-1 in Cx32 transfectants. These results suggest that Cx32 expression is in part related to induction of tight junctions through modulation of MAGI-1 expression in an immortalized mouse hepatic cell line.

Actins↗
PubMed2004

Thr(207) of claudin-5 is involved in size-selective loosening of the endothelial barrier by cyclic AMP.

We have recently shown that cyclic AMP (cAMP) increases claudin-5 immunoreactivity along cell boundaries and could promote phosphorylation of claudin-5 on threonine residues in porcine blood-brain barrier (BBB) endothelial cells via a protein kinase A (PKA)-dependent pathway (Exp. Cell Res. 290 [2003] 275). Along this line, we identified a putative phosphorylation site for PKA at Thr(207) in the intracytoplasmic carboxyl terminal domain of claudin-5. To clarify the biological significance of this site in regulation of endothelial barrier functions, we established rat lung endothelial (RLE) cells expressing doxycycline (Dox)-inducible wild-type claudin-5 and a mutant with a substitution of Ala for Thr(207) (CL5T207A). We show that induction of wild-type claudin-5 is sufficient to reconstitute the paracellular barrier against inulin (5 kDa), but not mannitol (182 Da), in leaky RLE cells. By contrast, the barrier against both molecules was induced in the mutant cells. We also demonstrate that, upon cAMP treatment, Thr(207) of claudin-5 is involved in enhancement of claudin-5 immunoreactive signals along cell borders, rapid reduction in transendothelial electrical resistance (TER), and loosening of the claudin-5-based endothelial barrier against mannitol, but not inulin. cAMP decreased the claudin-5-based endothelial barrier, strongly suggesting that other tight-junction molecule(s) are required to elevate endothelial barrier functions in response to cAMP.

Alanine↗