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Biomedical subjects

S Kitajima

Publications and source records attributed to S Kitajima.

At least 37 records · Page 2Linked to original sources

Dextran sodium sulfate-induced colitis in germ-free IQI/Jic mice.

This study presents a histological examination of dextran sodium sulfate (DSS)-induced colitis in germ-free (GF) mice. A comparison of the pathology between GF and conventionalized mice (CVz) was made to determine the role that intestinal microflora play in DSS-induced colitis. To induce colitis, GF and CVz IQI/Jic mice were given either 5% or 1% DSS orally. Administration of 5% DSS, a common concentration used to induce colitis in mice, caused gross rectal bleeding and a marked decrease in hematocrit as early as day one in GF mice. These mice died on day three due to massive bleeding into the intestinal lumen. In contrast, CVz mice did not die during the seven-day experimental period. Histopathological examination three days after administration of 5% DSS did not reveal any colitis lesions in GF mice, but CVz mice had developed moderate colitis in the large intestine. Administration of a low concentration of DSS (1%), which only induces mild basal crypt loss in CVz mice, caused severe colitis in the distal colon in GF mice, and they died on day 14. These data suggest that intestinal microflora are not necessary for the induction of colitis. Furthermore, DSS may be highly toxic to GF mice, and when given at a concentration of 5% it causes massive bleeding into the intestinal lumen resulting in death prior to development of colitis.

Administration, Oral↗

Manipulation of the somatosensory cortex modulates stimulus-induced repetitive ear movements in a seizure-sensitive strain of gerbil.

Some Mongolian gerbils (Meriones unguiculatus) respond to stimulation by seizures, the pattern of which changes progressively during development. We previously established a seizure-sensitive strain, MGS/Idr, in which all animals exhibit such stimulus-induced seizures. We have now noted that all adults of this strain also show repetitive backward movements of the ears at the ears at the beginning of stimulus-induced seizures, although the incidence varies with the individual. We examined whether the cerebral cortex was involved in these movements and found that electrical stimulation of an area of the somatosensory cortex elicited strong backward movement of the ear on the contralateral side, and that unilateral application of bicuculline, a GABAA receptor antagonist, induced spontaneous repetitive backward movements of the same ear. In this area, sharp waves appeared in the electrocortigram during the repetitive ear movements induced by seizure-inducing stimuli. Unilateral ablation of this area abolished stimulus-induced repetitive movements of the contralateral ear, but had no effects on those of the ipsilateral ear. These results suggest that, in certain types of seizure-susceptible subjects, it may be possible to modify stimulus-induced repetitive movements by manipulating a certain area of the somatosensory cortex which is related to these movements and that this gerbil strain may be useful in research on this subject.

Animals↗

Glial cells contribute more to iron and aluminum accumulation but are more resistant to oxidative stress than neuronal cells.

Iron (Fe) and aluminum (Al) have been implicated in the pathogenesis of Alzheimer's disease (AD). In this study, we examined neuronal and glial cells to clarify which contributes most to metal accumulation after internalization through the transferrin-independent iron uptake (Tf-IU) systems in primary neuronal and glial predominant (NP and GP) cells from rat cerebral cortex, which affect the accumulation of transition metals in a variety of cultured cells. Al more significantly upregulated the Tf-IU activity in GP cells than in NP cells. GP cells were more resistant to Fe and Al exposure than NP cells. However, a chemiluminescence analysis specific for reactive oxygen species (ROS) showed that ROS levels in Fe- or Al-loaded NP cells were twice as high as in Fe- or Al-loaded GP cells. Northern blot analysis and gel retardation assay showed that the Al and Fe exposure taken up by the cells suppress Tf receptor mRNA expression to a greater extent in GP than NP cells, indicating that Al and Fe more markedly accumulate in glial than in neuronal cells. These results suggest that glial cells rather than neuronal cells contribute to the metal accumulation and are more resistant to oxidative stress caused by metals than neuronal cells. The present study may help to explain the pathogenesis of neurodegeneration in AD disorders caused by metal-generated oxidative stress.

Aluminum↗

Drosophila von Hippel-Lindau tumor suppressor complex possesses E3 ubiquitin ligase activity.

Mutations of the von Hippel-Lindau (VHL) tumor suppressor gene predispose individuals to a variety of human tumors, including renal cell carcinoma, hemangioblastoma of the central nervous system, and pheochromocytoma. Here we report on the identification and characterization of the Drosophila homolog of VHL. The predicted amino acid sequence of Drosophila VHL protein shows 29% identity and 44% similarity to that of human VHL protein. Biochemical studies have shown that Drosophila VHL protein binds to Elongins B and C directly, and via this Elongin BC complex, associates with Cul-2 and Rbx1. Like human VHL, Drosophila VHL complex containing Cul-2, Rbx1, Elongins B and C, exhibits E3 ubiquitin ligase activity. In addition, we provide evidence that hypoxia-inducible factor (HIF)-1alpha is the ubiquitination target of both human and Drosophila VHL complexes.

Amino Acid Sequence↗

Homocysteine-responsive ATF3 gene expression in human vascular endothelial cells: activation of c-Jun NH(2)-terminal kinase and promoter response element.

Activating transcription factor (ATF) 3 is a member of ATF/cyclic adenosine monophosphate (cAMP)-responsive element binding protein (ATF/CREB) family of transcription factors and functions as a stress-inducible transcriptional repressor. To understand the stress-induced gene regulation by homocysteine, we investigated activation of the ATF3 gene in human endothelial cells. Homocysteine caused a rapid induction of ATF3 at the transcriptional level. This induction was preceded by a rapid and sustained activation of c-Jun NH(2)-terminal kinase/stress-activated protein kinase (JNK/SAPK), and dominant negative mitogen-activated protein kinase kinase 4 and 7 abolished these effects. The effect of homocysteine appeared to be specific, because cysteine or homocystine had no appreciable effect, but it was mimicked by dithiothreitol and beta-mercaptoethanol as well as tunicamycin. The homocysteine effect was not inhibited by an active oxygen scavenger. Deletion analysis of the 5' flanking sequence of the ATF3 gene promoter revealed that one of the major elements responsible for the induction by homocysteine is an ATF/cAMP responsive element (CRE) located at -92 to -85 relative to the transcriptional start site. Gel shift, immunoprecipitation, and cotransfection assays demonstrated that a complex (or complexes) containing ATF2, c-Jun, and ATF3 increased binding to the ATF/CRE site in the homocysteine-treated cells and activated the ATF3 gene expression, while ATF3 appeared to repress its own promoter. These data together suggested a novel pathway by which homocysteine causes the activation of JNK/SAPK and subsequent ATF3 expression through its reductive stress. Activation of JNK/SAPK and ATF3 expression in response to homocysteine may have a functional role in homocysteinemia-associated endothelial dysfunction.

Activating Transcription Factor 3↗

Repression of TNF-alpha-induced E-selectin expression by PPAR activators: involvement of transcriptional repressor LRF-1/ATF3.

Peroxisome proliferator-activated receptor (PPAR) activators were shown to inhibit the expression of E-selectin of human vascular endothelial cells in response to tumor necrosis factor-alpha (TNF-alpha). Troglitazone, pioglitazone, alpha-clofibrate, and 15-deoxy-Delta12,14-prostaglandin J2 all inhibited the TNF-alpha-stimulated E-selectin gene transcription in reporter assay. To further clarify the underlying transcriptional regulation, nuclear factor(s) that binds to the nuclear factor-endothelial leukocyte adhesion molecule 1 (NF-ELAM1) site of the E-selectin gene promoter was investigated. The activators caused a significant induction of liver regenerating factor 1 (LRF1)/activating transcription factor 3 (ATF3), which bound to the NF-ELAM1 site and repressed the TNF-alpha-induced E-selectin gene expression. From these data, the effect of PPAR activators was mediated, in part, through the induction of LRF1/ATF3. This might provide a novel molecular mechanism of anti-inflammatory effect of PPAR activators.

Activating Transcription Factor 3↗

Identification and characterization of Elongin A2, a new member of the Elongin family of transcription elongation factors, specifically expressed in the testis.

The Elongin complex stimulates the rate of transcription elongation by RNA polymerase II by suppressing the transient pausing of the polymerase at many sites along the DNA template. Elongin is composed of a transcriptionally active A subunit and two small regulatory B and C subunits, the latter of which bind stably to each other to form a binary complex that interacts with Elongin A and strongly induces its transcriptional activity. To further understand the roles of Elongin in transcriptional regulation, we attempted to identify Elongin-related proteins. Here, we report on the cloning, expression, and characterization of human Elongin A2, a novel transcription elongation factor that exhibited 47% identity and 61% similarity to Elongin A. Biochemical studies have shown that Elongin A2 stimulates the rate of transcription elongation by RNA polymerase II and is capable of forming a stable complex with Elongin BC. However, in contrast to Elongin A, its transcriptional activity is not activated by Elongin BC. Northern blot analysis revealed that Elongin A2 mRNA was specifically expressed in the testis, suggesting that Elongin A2 may regulate the transcription of testis-specific genes.

Amino Acid Sequence↗

Time course of ultrastructural changes and immunoelectron microscopic localization of neurocalcin in motor endplates of the lumbrical muscles of rats given a single administration of 2,5-di(tert-butyl)-1,4-hydroquinone.

A time-course study of ultrastructural changes and immunoelectron microscopic localization of neurocalcin was performed on motor endplates of the lumbrical muscles of female Wistar rats given a single oral administration of 2,5-di(tert-butyl)-1,4-hydroquinone (DTBHQ) at a dose of 120 mg/kg. Toxic signs such as salivation and muscle weakness of the hind legs appeared from 3 h after DTBHQ administration. No remarkable macroscopic or light microscopic changes were noted in the lumbrical muscles of the treated rats. At the ultrastructural level, neurotoxicity characterized by a decreases or loss of synaptic vesicles and mitochondria was observed after 24 h and at the 1-week time point, nerve endings had disappeared in some of the motor endplates, while many neurite nerve endings suggestive of early stage regeneration were apparent. After 6 weeks, newly formed reinnervated endplates were observed. Immunoelectron microscopically, the synaptic vesicle membranes were heavily labeled for neurocalcin in the control rats, but not at 24 h after DTBHQ treatment. Synaptic vesicle membranes in the DTBHQ group were weakly labeled at 1 week, but strongly at 6 weeks. The results strongly suggest that DTBHQ targets the motor endplates in the rat lumbrical muscles, causing depletion of neurocalcin in the synaptic vesicles followed by their loss.

Animals↗

The adaptive response of transforming growth factor-beta 2 and -beta RII in the overloaded, regenerating and denervated muscles of rats.

Using a muscle cell line and satellite cell cultures, it has been shown that transforming growth factor-beta (TGF-beta) has a powerful inhibitory effect on myoblast replication and differentiation. However, little work has been done on the possible role of TGF-beta in adult muscle in vivo. Using Western blot and immunohistochemical analyses, we investigated normal distribution of TGF-beta 2 and TGF-beta RII proteins between slow and fast-type muscles, and the adaptive response of these proteins in the mechanically overloaded muscles, in the regenerating muscles following bupivacaine injection and in the denervated muscle after section of sciatic nerve. Slight TGF-beta 2 immunoreactivity was detected both in slow- and fast-type muscles of mature rat. The amount of TGF-beta RII protein was markedly greater in fast-type muscles. In the overloaded muscle, immunohistochemical analysis showed a marked increase in TGF-beta 2 immunoreactivity in the mononuclear cells (probably endothelial and perithelial or smooth muscle cells of endomysial capillaries) of the extracellular space at 3 and 6 days post surgery. Rapid increase of TGF-beta 2 protein and concomitant decrease of the receptor (TGF-beta RII) were observed in the mechanically overloaded and regenerating muscles. On the other hand, denervation of slow- and fast-type muscles showed a rapid increase in TGF-beta 2 protein, but did not elicit a concomitant decrease of TGF-beta RII. These results indicate that TGF-beta RII is preferentially distributed in fast-type muscles. Furthermore, TGF-beta 2 may play an important role in muscle hypertrophy and regeneration by the usage of TGF-beta RII.

Animals↗

Mesp1 expression is the earliest sign of cardiovascular development.

Understanding the molecular mechanism leading to formation of the heart and vasculature during embryogenesis is critically important because malformation of the cardiovascular system is the most frequently occurring type of birth defect. While the hearts of all vertebrates are derived from bilateral paired fields of primary mesodermal cells that are specified to the cardiac lineage during gastrulation, the mechanism for lineage restriction, and the origin of the myocardium and endocardium have not been defined. Recently, we found that a transcription factor, Mesp1, is expressed in almost all precursors of the cardiovascular system and plays an essential role in cardiac morphogenesis. Mesp1 may play a key role in the early specification for cardiac precursor cells.

Animals↗

Mesp2 initiates somite segmentation through the Notch signalling pathway.

The Notch-signalling pathway is important in establishing metameric pattern during somitogenesis. In mice, the lack of either of two molecules involved in the Notch-signalling pathway, Mesp2 or presenilin-1 (Ps1), results in contrasting phenotypes: caudalized versus rostralized vertebra. Here we adopt a genetic approach to analyse the molecular mechanism underlying the establishment of rostro-caudal polarity in somites. By focusing on the fact that expression of a Notch ligand, Dll1, is important for prefiguring somite identity, we found that Mesp2 initiates establishment of rostro-caudal polarity by controlling two Notch-signalling pathways. Initially, Mesp2 activates a Ps1-independent Notch-signalling cascade to suppress Dll1 expression and specify the rostral half of the somite. Ps1-mediated Notch-signalling is required to induce Dll1 expression in the caudal half of the somite. Therefore, Mesp2- and Ps1-dependent activation of Notch-signalling pathways might differentially regulate Dll1 expression, resulting in the establishment of the rostro-caudal polarity of somites.

Animals↗

Characterization of gene expression of NsERFs, transcription factors of basic PR genes from Nicotiana sylvestris.

Three genes of NsERFs (EREBPs), transcription factors for GCC box of basic PR genes, were isolated from Nicotiana sylvestris. Analyses of transgenic tobacco carrying the NsERF promoter::GUS genes showed that expression of all NsERF genes in leaves was induced by ethylene. Sequence analyses indicated that the 5'-upstream region of NsERF3 gene has the GCC box. In contrast, the promoter regions of NsERF2 and 4 have no GCC box, whereas NsERF2 has a putative EIN3 binding site. Tissue/cell specific expression is also discussed.

Amino Acid Sequence↗

MesP1 and MesP2 are essential for the development of cardiac mesoderm.

The transcription factors, MesP1 and MesP2, sharing an almost identical bHLH motif, have an overlapping expression pattern during gastrulation and somitogenesis. Inactivation of the Mesp1 gene results in abnormal heart morphogenesis due to defective migration of heart precursor cells, but somitogenesis is not disrupted because of normal expression of the Mesp2 gene. To understand the cooperative functions of MesP1 and MesP2, either a deletion or sequential gene targeting strategy was employed to inactivate both genes. The double-knockout (dKO) embryos died around 9.5 days postcoitum (dpc) without developing any posterior structures such as heart, somites or gut. The major defect in this double-knockout embryo was the apparent lack of any mesodermal layer between the endoderm and ectoderm. The abnormal accumulation of cells in the primitive streak indicates a defect in the migratory activity of mesodermal cells. Molecular markers employed to characterize the phenotype revealed a lack of the cranio-cardiac and paraxial mesoderm. However, the axial mesoderm, as indicated by brachyury (T) expression, was initially generated but anterior extension was halted after 8.5 dpc. Interestingly, a headfold-like structure developed with right anterior-posterior polarity; however, the embryos lacked any posterior neural properties. The persistent and widely distributed expression of Cerberus-like-1(Cer1), Lim1 and Otx2 in the anterior endoderm might be responsible for the maintenance of anterior neural marker expression. We also performed a chimera analysis to further study the functions of MesP1 and MesP2 in the development of mesodermal derivatives. In the chimeric embryos, dKO cells were scarcely observed in the anterior-cephalic and heart mesoderm, but they did contribute to the formation of the somites, notochord and gut. These results strongly indicate that the defect in the cranial-cardiac mesoderm is cell-autonomous, whereas the defect in the paraxial mesoderm is a non-cell-autonomous secondary consequence.

Animals↗

Histological analysis of murine colitis induced by dextran sulfate sodium of different molecular weights.

In this study, we examined the relationship between the molecular weight of dextran sulfate sodium (DSS) and the features of colitis in a DSS-induced mouse model of human ulcerative colitis. DSS at three different molecular weights, 5 kD, 40 kD and 500 kD, was used in this study. DSS was administered in drinking water at 5% (w/v) to 6-7-week-old female BALB/c mice. After 7 days of treatment with DSS, the large intestine was examined histopathologically. Colitis was characterized by a loss of crypts, infiltration of inflammatory cells into the mucosa and submucosa, edema of the submucosa, erosion and ulceration and was observed in mice given the 5 kD and 40 kD forms but not the 500 kD. In the 5 kD group, colitis was observed predominantly in the cecum and upper colon. Colitis in the 40 kD group was more severe than that in the 5 kD group, and in the 40 kD group it was more severe in the lower colon than in the upper colon. These findings suggest the molecular weight of DSS to be an important factor in the murine model of colitis.

Animals↗

Studies on thyroid function in rats subjected to repeated oral administration with kojic acid.

To elucidate the effects of kojic acid on thyroid function, the compound was given orally to male rats for 4 weeks at 0, 4, 15, 62.5, 250 and 1,000 mg/kg. In 1,000 mg/kg treatment of kojic acid, the rats showed a slight decrease in motility, inhibition of body weight gain, and a decrease in food consumption. An increase in thyroid weight and a morphological change, i.e., hypertrophy of epithelial cells of the thyroid gland follicles, were observed after 1 week of administration. In addition, the uptake of radioactive iodine from blood into the thyroid gland was enhanced and the TCA-precipitable radioactive iodine in the thyroid gland increased in those rats. However, the rates of the iodination in the thyroid gland did not change during the experiment period. Although serum T4 concentration was low in the rats treated with 1,000 mg/kg kojic acid, it was not observed in any changes in TSH concentration. None of these changes were found in the other groups. These observations suggest that massive administration of kojic acid may decrease blood T4 concentration and that thyroid function may be enhanced compensatorily. On the other hand, the absorption of kojic acid was rapid as manifested by the Tmax of blood concentrations of radioactivity, which was as short as 1.0 +/- 0.0 hr, and the t1/2 was 4.8 +/- 0.3 hr. Blood concentrations of radioactivity disappeared nearly completely at 24 hr after administration. This result indicates that the toxic effect observed on the thyroid gland treated with only the largest dosage of kojic acid may depend on a fast decrease following a transient increase of concentration of the compound in the blood.

Animals↗

Immunohistochemical estimation of in-situ cell cycle time in neoplastic epithelial cells in human large intestine: a new cell proliferation index.

In-situ cell cycle time (in-situ Tc) of epithelial cells could be estimated by using a formula; in-situ Tc = cell proliferation rate divided by mitosis rate, on a scale of Tm (cell cycle time in M phase) arbitrary unit (AU), In order to see the nature of in-situ Tc in the adenoma-carcinoma sequence in the human large intestine, the in-situ Tc in 27 cases of adenoma and 71 cases of adenocarcinoma with adenoma components in the human large intestine was estimated by using this formula, counting proliferating cells and mitotic cells in the immunohistochemistry of Ki-67 antigen. C12 antigen was examined as an oncogenic progression indicator in the adenoma-carcinoma sequence. The in-situ Tc tended to shorten in adenoma in accordance with the histological grading of atypia but not in adenoma component. No significant differences in the in-situ Tc was recognized as a whole among adenomas, adenoma components and adenocarcinomas in the mucosa, whereas the in-situ Tc of adenoma components with moderate to severe atypia was significantly longer than that of adenocarcinomas in the mucosa (p = 0.05). The in-situ Tc lengthened in adenocarcinomas invading the submucosa and shortened in adenocarcinomas invading the proper muscular layer. The cases expressing the C12 antigen increased in order of adenoma, adenoma component and adenocarcinoma. The cases expressing the C12 antigen indicated short in situ Tc in the adenomas and adenocarcinomas but not in the adenoma components. Thus, the estimated in-situ Tc is a useful index of the oncogenetic progression, which is different from that detected by the C12 antigen.

Adenocarcinoma↗