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Kenji Watanabe

Publications and source records attributed to Kenji Watanabe.

At least 37 records · Page 2Linked to original sources
PubMed2005

Maintaining the redox-balance intact: gosha-jinki-gan but not insulin activates retinal soluble guanylate cyclase in diabetic rats.

Strategies to prevent hyperglycemia-induced cytotoxic reactive oxygen species in the retina include the prevention of free radical production, activation of radical-scavenging capacities and inhibition of aldose reductase. This study examined the effect of the standardized Japanese herbal extract product gosha-jinki-gan (GJG) in comparison to insulin treatment in the rat retina. Diabetes was induced in male Wistar rats by single injection of streptozotocin (50 mg/kg i.p.). At 6 and 12 weeks, eye-cups were removed for immunohistochemistry. At 12 weeks, lipid peroxidation (tested with the antiacrolein antibody, Ab5F6) was enhanced significantly in the untreated diabetic group. This effect was absent in both treatment groups, notably in the outer retina. A similar result was obtained for nitrotyrosine overproduction. As an early treatment effect, GJG -- but not insulin -- enhanced soluble guanylate cyclase (sGC) activation (using the function-sensing antibody, MoAb 3221). GJG not only reduces nitroxidative stress and lipid peroxidation in the retina, it also ameliorates glucose metabolism within the cells. We propose that the high glucose turnover in the insulin-treated model disturbs the intracellular redox equilibrium, one result of which might be the impaired sGC activation.

Animals↗
PubMed2005

alpha-Glucosidase from a strain of deep-sea Geobacillus: a potential enzyme for the biosynthesis of complex carbohydrates.

An alpha-glucosidase from Geobacillus sp. strain HTA-462, one of the deepest sea bacteria isolated from the sediment of the Mariana Trench, was purified to homogeneity and estimated to be a 65-kDa protein by SDS-PAGE. At low ion strength, the enzyme exists in the homodimeric form (130 kDa). It is a thermo- and alkaline-stable enzyme with a half-life of 13.4 h and a maximum hydrolytic activity at 60 degrees C and pH 9.0 in 15 mM glycine-NaOH buffer. The enzyme exclusively hydrolyzed alpha-1,4-glycosidic linkages of oligosaccharides in an exo-type manner. The enzyme had an overwhelming transglycosylation activity and glycosylated various non-sugar molecules when maltose was used as a sugar donor. It converted maltose to isomaltose. The gene encoding the enzyme was cloned and sequenced. The recombinant enzyme could be extracellularly overproduced by Bacillus subtilis harboring its gene and preserved the primary properties of the native enzyme. Site-directed mutagenesis experiments showed that Asp98 is essential for the enzyme activity in addition to Asp199, Asp326, and Glu256.

Amino Acid Sequence↗
PubMed2005

Potassium currents in isolated statocyst neurons and RPeD1 in the pond snail, Lymnaea stagnalis.

To begin to determine the underlying neural mechanisms of memory formation, we studied two different cell types that play important roles in different forms of associative learning in Lymnaea. Statocyst neurons (hair cells) mediate classical conditioning, whereas RPeD1 is a site of memory formation induced by operant conditioning of aerial respiration. Because potassium (K(+)) channels play a critical role in neuronal excitability, we initiated studies on these channels in the aforementioned neurons. Three distinct K(+) currents are expressed in the soma of both the hair cells and RPeD1. In hair cells and RPeD1, there is a fast activating and rapidly inactivating 4-aminopyridine (4-AP)-sensitive A current (I(A)), a tetraethyl ammonium (TEA)-sensitive delayed rectifying current, which exhibits slow inactivation kinetics (I(KV)), and a TEA- and 4-AP-insensitive Ca(2+)-dependent current (I(Ca-K)). In hair cells, the activation voltage of I(A); its half-maximal steady-state activation voltage and its half-maximal steady-state inactivation were at more depolarized levels than in RPeD1. The time constant of recovery from I(A) inactivation was slightly faster in hair cells. I(A) in hair cells is also smaller in amplitude than in RPeD1 and is activated at more depolarized potentials. In like manner, I(KV) is smaller in hair cells and is activated at more depolarized potentials than in RPeD1.

4-Aminopyridine↗
PubMed2005

Skin temperature rise induced by calcitonin gene-related peptide in gonadotropin-releasing hormone analogue-treated female rats and alleviation by Keishi-bukuryo-gan, a Japanese herbal medicine.

The effects of Keishi-bukuryo-gan on calcitonin gene-related peptide (CGRP)-induced elevation of skin temperature were investigated in gonadotropin-releasing hormone (GnRH) analogue-treated female rats. Leupline (1.0 mg/kg) as the GnRH analogue was subcutaneously (s.c.) injected into female rats. After Keishi-bukuryo-gan (100-1,000 mg/kg, p.o.) or 17beta-estradiol (0.010 mg/kg, s.c.) was administered to GnRH analogue-treated rats for 14 days, CGRP-induced skin temperature elevation, concentration of plasma 17beta-estradiol and pituitary gonadotropin (luteinizing hormone; LH, and follicle stimulating hormone; FSH) were measured. In addition, effects of 17beta-estradiol and Keishi-bukuryo-gan on the proliferation of estrogen-dependent human breast cancer (MCF-7) cells were investigated under in vitro conditions. GnRH analogue significantly lowered the concentrations of plasma 17beta-estradiol and pituitary gonadotropins. Tissue weights of the ovaries and uterus were also decreased by the analogue. Under the condition of estrogen deficiency, intravenous (i.v.) injection of exogenous CGRP (10 microg/kg) elevated the skin temperature of the hind paws more significantly than it did in sham-treated control rats. Estrogen supplementation inhibited this elevation of skin temperature with restoration of both the lowered plasma estrogen level and the decreased uterine weight in GnRH analogue-treated rats. On the other hand, Keishi-bukuryo-gan inhibited the elevation of skin temperature in a dose-dependent manner without restoring the plasma estrogen level and uterine weight. In addition, in an in vitro study, MCF-7 cells proliferated in a dose-dependent manner by the addition of 17beta-estradiol (10(-13)-10(-8) M) to the medium. However, Keishi-bukuryo-gan (10(-6)-10(-4) mg/ml) did not activate the MCF-7 cell proliferation. These results suggest that Keishi-bukuryo-gan, which does not exhibit estrogen activity, may be useful for the treatment of hot flashes in women who are undergoing medical ovariectomy with a GnRH analogue.

Animals↗
PubMed2005

Characterization of tryptic hydrolysis of alpha-lactalbumin/saponin mixture and structural change of alpha-lactalbumin interacting with soybean saponin.

Bovine milk alpha-lactalbumin (alpha-La) was mixed with soybean saponin, and the resulting mixture was hydrolyzed by trypsin. Saponin increased the tryptic-hydrolysis level of alpha-La only at relatively high phosphate buffer concentrations (> or = 0.05 M). T(1) experiments with acetylated soybean saponin demonstrated that there were some interactions between alpha-La and saponin not only at high concentrations of phosphate buffers but even at low concentrations as well. Circular dichroism spectra of alpha-La showed that the tertiary structure of alpha-La was changed through interactions with saponin only at high buffer concentrations. Furthermore, by analyzing the tryptic peptides from an alpha-La/saponin mixture, hydrolyzing rates at all or some of K5, R10, and K16 of alpha-La were accelerated by saponin interactions. The increase in the tryptic hydrolysis of alpha-La by saponin addition was considered due to modification of the tertiary structure of alpha-La by saponin.

Animals↗
PubMed2005

Distribution of the stem cells (neoblasts) in the planarian Dugesia japonica.

It has been postulated that the high regeneration ability of planarians is supported by totipotent stem cells, called neoblasts. There have been a few reports showing the distribution of neoblasts in planarians. However, the findings were not completely consistent. To determine the distribution of neoblasts, we focused on proliferating cell nuclear antigen (PCNA), which is present in proliferative cells. We cloned and sequenced the cDNA of PCNA from the planarian Dugesia japonica and produced an antiserum recognizing the gene product. X-ray irradiation caused rapid loss of all PCNA-positive cells and loss of the neoblasts (which were morphologically defined by the presence of the chromatoid body), strongly suggesting that all PCNA-positive cells were true neoblasts. Using the antiserum, we were successful in identifying the neoblasts more clearly than any previous work. In addition to their dispersed distribution in the dorsal and ventral mesenchyme, the neoblasts were distributed as clusters along the midline and bilateral lines in the dorsal mesenchyme. We also examined the behavior of the neoblasts after decapitation. Decapitation did not seem to affect the migration of neoblasts far from the wound. We demonstrated here that DjPCNA is a powerful tool for identifying planarian neoblasts.

Amino Acid Sequence↗
PubMed2005

The herbal medicine Sho-saiko-to selectively inhibits CD8+ T-cell proliferation.

Sho-saiko-to (SST), a Chinese/Japanese traditional herbal medicine, has been widely used to treat chronic hepatitis in Japan, and the immunomodulatory properties of SST are likely to mediate its beneficial effect. In the present study, we examined the effects of SST and its various ingredients on the count and proliferation of T-cell subsets in cultured splenocytes and hepatic mononuclear cells. SST, wogonin-7-O-glucuronoside (a major SST ingredient), and wogonin (an intestinal metabolite of wogonin-7-O-glucuronoside) increased CD4/CD8 ratio via a decrease of CD8+ T-cell counts with no effect on CD4+ T-cell counts. Flow cytometric analyses of viability, proliferation, and cell cycle revealed that wogonin suppressed CD8+ T-cell proliferation without inducing cell death. SST and wogonin administered to mice increased the CD4/CD8 ratio in hepatic mononuclear cells but not in splenocytes. These findings suggest that SST may modulate the CD4/CD8 ratio via the selective inhibition of CD8+ T-cell proliferation by the SST ingredient wogonin-7-O-glucuronoside or its metabolite wogonin.

Animals↗
PubMed2005

Macroarray-based analysis of tail regeneration in Xenopus laevis larvae.

Xenopus larvae possess a remarkable ability to regenerate their tails after they have been severed. To gain an understanding of the molecular mechanisms underlying tail regeneration, we performed a cDNA macroarray-based analysis of gene expression. A Xenopus cDNA macroarray representing 42,240 independent clones was differentially hybridized with probes synthesized from the total RNA of normal and regenerating tails. Temporal expression analysis revealed that the up-regulated genes could be grouped into early or late responding genes. A comparative expression analysis revealed that most genes showed similar expression patterns between tail development and regeneration. However, some genes showed regeneration-specific expression. Finally, we identified 48 up-regulated genes that fell into several categories based on their putative functions. These categories reflect the various processes that take place during regeneration, such as inflammation response, wound healing, cell proliferation, cell differentiation, and control of cell structure. Thus, we have identified a panel of genes that appear to be involved in the process of regeneration.

Animals↗
PubMed2005

A Kampo formula Juzen-taiho-to induces expression of metallothioneins in mice.

Juzen-taiho-to (JTX), one of the commonly prescribed traditional Japanese herbal medicines (Kampo), is indicated for adjunctive treatment of cancers and autoimmune diseases. To understand the mechanisms underlying the clinical effects of JTX, the effects of orally administered JTX on the expression of metallothioneins (MTs) were examined in the liver, spleen, small and large intestines of mice. In addition, the expression of MTs in specific pathogen free (SPF) mice was examined to understand the participation of intestinal bacteria in the expression of MTs. JTX enhanced expression of MT-I and -II significantly in the liver of SPF mice. Induction of MT-II expression was observed also in the small intestine. Intestinal bacteria appeared to have no effect on MTs expression. Neither expression of MT-III nor its induction was observed in any tissue. These findings strongly suggest that MTs should mediate at least some effects of JTX in mice.

Administration, Oral↗
PubMed2005

Identification of asymmetrically localized transcripts along the animal-vegetal axis of the Xenopus egg.

In many organisms, proper embryo development depends on the asymmetrical distribution of mRNA in the cytoplasm of the egg. Here we report comprehensive screening of RNA localized in the animal or vegetal hemisphere of the Xenopus egg. Macroarrays including over 40,000 independent embryonic cDNA clones, representing at least 17,000 unigenes, were differentially hybridized with labeled probes synthesized from the mRNA of animal or vegetal blastomeres. After two rounds of screening, we identified 33 clones of transcripts that may be preferentially distributed in the vegetal region of the early stage embryo, but transcripts localized in the animal region were not found. To assess the array results, we performed northern blot and quantitative real-time reverse transcription-polymerase chain reaction analysis. As a result, 21 transcripts of the 33 were confirmed to be localized in the vegetal region of the early stage embryo. Whole-mount in situ hybridization analysis revealed that 11 transcripts, including 7 previously reported genes, were localized in the vegetal hemisphere of the egg. These 11 transcripts were categorized into three groups according to their expression patterns in the egg. The first group, which contained four transcripts, showed uniform expression in the vegetal hemisphere, similar to VegT. The second group, which contained three transcripts, showed gradual expression from the vegetal pole to the equator, similar to Vg1. The last group, which contained three transcripts, was expressed at the germ plasm, similar to Xdazl. One transcript, Xwnt11, showed both the second and the third expression patterns.

Animals↗
PubMed2005

Planarian peptidylglycine-hydroxylating monooxygenase, a neuropeptide processing enzyme, colocalizes with cytochrome b561 along the central nervous system.

Planarians are one of the simplest animal groups with a central nervous system. Their primitive central nervous system produces large quantities of a variety of neuropeptides, of which many are amidated at their C terminus. In vertebrates, peptide amidation is catalyzed by two enzymes [peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxylglycine alpha-amidating lyase] acting sequentially. In mammals, both enzymatic activities are contained within a single protein that is encoded by a single gene. By utilizing PCR with degenerate oligonucleotides derived from conserved regions of PHM, we succeeded in cloning a full-length cDNA encoding planarian PHM. The deduced amino acid sequence showed full conservation of five His residues and one Met residue, which bind two Cu atoms that are essential for the activity of PHM. Northern blot analysis confirmed the expression of a PHM mRNA of the expected size. Distribution of the mRNA was analyzed by in situ hybridization, showing specific expression in neurons with two morphologically distinct structures, a pair of the ventral nerve cords and the brain. The distribution of PHM was very similar to that of cytochrome b561. This indicates that the ascorbate-related electron transfer system operates in the planarian central nervous system to support the PHM activity and that it predates the emergence of Plathelminthes in the evolutionary history.

Amino Acid Sequence↗
PubMed2005

Dislocation of tight junction proteins without F-actin disruption in inactive Crohn's disease.

Crohn's disease is associated with increased permeability of the intestinal barrier even in quiescent patients. Increased intestinal permeability may cause dysregulated immunological responses in the intestinal mucosa that leads to chronic intestinal inflammation. We have studied the expression of tight junction proteins (occludin and zonula occludens), alpha2-smooth muscle actin, TGF-beta with a cytoskeletal protein (F-actin) in the intestinal epithelium of patients with inflammatory bowel disease. Surgical samples were obtained from 6 controls (individuals without inflammatory bowel disease), 8 patients with ulcerative colitis and 7 patients with Crohn's disease. F-actin was visualized with fluorescein phalloidin. Tight junction proteins, alpha2 smooth muscle actin, and TGFbeta were visualized by the immunofluorescent method. Occludin and zonula occludens found in apical tight junctions in normal epithelium were dislocated to the basolateral position and in the lamina propria extracellular matrix in patients with Crohn's disease, while the structure of F-actin was maintained in inactive or minimally inflamed mucosa. TGF-beta positive inflammatory cells were increased in ulcerative colitis and Crohn's disease mucosa. Subepithelial myofibroblasts were constitutively found in controls, ulcerative colitis, and Crohn's disease mucosa. Latent dislocation of tight junction proteins, without disturbance of the cytoskeleton in the inactive mucosa of patients with Crohn's disease, may permit the invasion of gut antigens because the functional disruption of tight junctions could initiate an altered immune response.

Actins↗
PubMed2005

Significance of increased proliferation of immature plasma cells in the appendix of patients with ulcerative colitis.

The etiology of ulcerative colitis (UC) is not known. Recent studies support a primary role of the appendix in the pathogenesis of UC, however phenotypical studies of proliferating cells in the appendix have not been reported. We report phenotypical studies of lymphocytes and of proliferating subpopulations in the appendix of patients with inflammatory bowel disease and of controls. Surgical samples of the appendix were obtained from 5 patients with colon cancer, 5 with acute appendicitis, 12 with UC and 7 with Crohn's disease (CD). Frozen sections were cut from fixed samples, and immunostained with lymphocyte markers and anti-Ki-67 antibodies. The number of Ki-67(+) proliferating cells, CD19, and CD138 cells was significantly higher in the appendix of patients with UC than in controls, patients with acute appendicitis, and patients with CD. Immunohistological double staining revealed significant proliferation of CD3, CD19, and CD138 cells in the appendix of patients with UC. The proportions of Ki-67(+) cells in CD3, CD19, and CD138 cells were significantly higher in both total UC patients and patients in remission-stage UC, than in controls, patients with acute appendicitis, and patients with CD. Lamina propria cells in the appendix of patients with UC showed augmented proliferation with increased numbers of CD19 and CD138 cells. The number of CD3 cells was not significantly increased, but the proportion of proliferating CD3 cells was increased. An increased proportion of Ki-67(+) cells in CD19 and CD138 cells represents proliferation of immature plasma cells in the appendix of patients with UC, and proliferation of such immature plasma cells was seen in both active- and remission-stage UC. Proliferation of immature plasma cells in the appendix of patients with UC suggests a primary role of humoral immune responses in the pathogenesis of UC.

Adolescent↗
PubMed2005

[Carcinogenesis in ulcerative colitis-associated colon cancer and surveillance colonoscopy].

It has been widely accepted that long-standing cases with ulcerative colitis, especially pancolitis have higher risk of complicating colitic cancer. Colitic cancer often appears as multi-focal invasive features with un-differentiated histology. Therefore, endoscopic diagnosis of precancerous or cancer associated dysplastic lesions. Surveillance colonoscopy has preventing effects for developing colitic cancer. In western world, annual surveillance colonoscopy with multiple stepwise has been adopted. In Japan, surveillance colonoscopy with targeted biopsies assisted by chromoendoscopy and/or magnifying endoscopy has proven to be efficacious in finding such lesions.

Colitis, Ulcerative↗
PubMed2005

[Extraintestinal complications in patients with ulcerative colitis].

Extraintestinal complications of patients with ulcerative colitis contribute to quality of life and mortality of the patients. Extraintestinal complications involve many organ systems. Skin, mucocutaneous, hepatobiliary, and joints complications are major extraintestinal complications. Some disorders correspond the activity of the colitis, other complications develop independently of the bowel inflammation. The purpose of this review is to update the clinicians concerning the etiology, clinical course and the therapy of the extraintestinal manifestations associated with ulcerative colitis.

Cholangitis, Sclerosing↗
PubMed2004

Molecular dynamics of STAT3 on IL-6 signaling pathway in living cells.

Signal transducer and activator of transcription 3 (STAT3) is a critical signal transducer of interleukin-6 (IL-6) signaling. To investigate the mobility and the dynamics of STAT3 complex on IL-6 signaling in living cells, we generated a chimeric gene consisting of STAT3 fused to enhanced green fluorescence protein, STAT3-GFP. STAT3-GFP was expressed in Hep3B cells and the dynamics of this protein were analyzed by fluorescence correlation spectroscopy. After IL-6 stimulation, STAT3 translocated from the cytoplasm to the nucleus, as shown previously. According to the analysis of STAT3 diffusion in stable transformants, the number of STAT3 molecules at the cytoplasmic membrane and in the cytoplasm decreased after IL-6 stimulation. In the nucleus, the diffusion speed of STAT3 complex strongly decreased after IL-6 stimulation. Furthermore, we found that STAT3 existed as a complex whose molecular weight was less than 400kDa before IL-6 addition. However, IL-6 stimulation induced the formation of STAT3 dimer as a megacomplex form whose molecular weight was more than 1MDa at the cytoplasm and a very slow diffusion complex in the nucleus.

Cell Line, Tumor↗