Shinichi Yoshimura

A basolateral sorting motif in the MICA cytoplasmic tail

The MHC class I chain-related MICA molecule is a stress-induced, highly polymorphic, epithelia-specific, membrane-bound glycoprotein interacting with the activating NK cell receptor NKG2D and/or gut-enriched Vδ1-bearing γδ T cells. We have previously reported the presence of a MICA transmembrane-encoded short-tandem repeat harboring a peculiar allele, A5.1, characterized by a frame shift mutation leading to a premature intradomain stop codon, thus denying the molecule of its 42-aa cytoplasmic tail. Given that this is the most common population-wide MICA allele found, we set out to analyze the functional consequences of cytoplasmic tail deletion. Here, we show native expression of MICA at the basolateral surface of human intestinal epithelium, the site of putative interaction with intraepithelial T and NK lymphocytes. We then demonstrate, in polarized epithelial cells, that although the full-length MICA protein is sorted to the basolateral membrane, the cytoplasmic tail-deleted construct as well as the naturally occurring A5.1 allele are aberrantly transported to the apical surface. Site-directed mutagenesis identified the cytoplasmic tail-encoded leucine-valine dihydrophobic tandem as the basolateral sorting signal. Hence, the physiological location of MICA within epithelial cells is governed by its cytoplasmic tail, implying impairment in A5.1 homozygous individuals, perhaps relevant to the immunological surveillance exerted by NK and T lymphocytes on epithelial malignancies.

Determination of nucleotide sequence of cDNA coding rat glutathione peroxidase and diminished expression of the mRNA in selenium deficient rat liver

The cDNA for rat glutathione peroxidase mRNA was isolated from liver cDNA library in lambda gt11 by cross-hybridization using the mouse cDNA, and its nucleotide sequence was determined. The selenocysteine which constitutes an active center of this enzyme was encoded by TGA, a nonsense codon in general, as was the cases with mouse and human glutathione peroxidase. Northern blot analysis elucidated that the mRNA for glutathione peroxidase was markedly diminished in selenium deficient rat liver as compared with that of normal rat livers. The result suggested that the de novo synthesis of the mRNA would be regulated by selenium.

Purification and immunohistochemical localization of rat liver glutathione peroxidase

Glutathione peroxidase was purified from the rat liver to give a single protein band in polyacrylamide gel electrophoresis. Rabbits were immunized with this purified enzyme, and a highly specific anti-glutathione peroxidase antiserum was obtained. Using this antibody, an immunohistochemical technique (the indirect method of peroxidase-labeled antibody) was applied to study the localization of the enzyme in the liver cells. On immunohistochemical observation, glutathione peroxidase was localized exclusively in the cytoplasm of hepatocytes, and a stronger immuno-staining was exhibited in the peripheries of the hepatic lobules than in the central zone.

New Fiber Formation in the Interstitial Spaces of Rat Skeletal Muscle During Postnatal Growth

The purpose of this study was to determine whether fiber hyperplasia occurs in the rat plantaris muscle during postnatal weeks 3–20. Total muscle fiber number, obtained via the nitric acid digestion method, increased by 28% during the early postnatal rapid growth phase (3–10 weeks), whereas the number of branched fibers was consistently low. Whole-muscle mitotic activity and amino acid uptake levels showed an inverse relationship to the increase in total fiber number. The expression of MyoD mRNA (RT-PCR) levels decreased from 3 to 20 weeks of age, as did the detection of anti-BrdU- and MyoD-positive cells in histological sections. Immunohistochemical staining patterns for MyoD, myogenin, or developmental myosin heavy chain on sections stained for laminin (identification of the basal lamina) and electron micrographs clearly indicate that de novo fiber formation occurred in the interstitial spaces. Myogenic cells in the interstitial spaces were negative for the reliable specific satellite cell marker M-cadherin. In contrast, CD34 (an established marker for hematopoietic stem cells)-positive cells were located only in the interstitial spaces, and their frequency and location were similar to those of MyoD- and/or myogenin-positive cells. These findings are consistent with fiber hyperplasia occurring in the interstitial spaces of the rat plantaris muscle during the rapid postnatal growth phase. Furthermore, these data suggest that the new fibers may be formed from myogenic cells in the interstitial spaces of skeletal muscle and may express CD34 that is distinct from satellite cells.

Major histocompatibility complex (Mhc) class Ib gene duplications, organization and expression patterns in mouse strain C57BL/6

BackgroundThe mouse has more than 30 Major histocompatibility complex (Mhc) class Ib genes, most of which exist in the H2 region of chromosome 17 in distinct gene clusters. Although recent progress in Mhc research has revealed the unique roles of several Mhc class Ib genes in the immune and non-immune systems, the functions of many class Ib genes have still to be elucidated. To better understand the roles of class Ib molecules, we have characterized their gene duplication, organization and expression patterns within the H2 region of the mouse strain C57BL/6.ResultsThe genomic organization of the H2-Q, -T and -M regions was analyzed and 21 transcribed Mhc class Ib genes were identified within these regions. Dot-plot and phylogenetic analyses implied that the genes were generated by monogenic and/or multigenic duplicated events. To investigate the adult tissue, embryonic and placental expressions of these genes, we performed RT-PCR gene expression profiling using gene-specific primers. Both tissue-wide and tissue-specific gene expression patterns were obtained that suggest that the variations in the gene expression may depend on the genomic location of the duplicated genes as well as locus specific mechanisms. The genes located in the H2-T region at the centromeric end of the cluster were expressed more widely than those at the telomeric end, which showed tissue-restricted expression in spite of nucleotide sequence similarities among gene paralogs.ConclusionDuplicated Mhc class Ib genes located in the H2-Q, -T and -M regions are differentially expressed in a variety of developing and adult tissues. Our findings form the basis for further functional validation studies of the Mhc class Ib gene expression profiles in specific tissues, such as the brain. The duplicated gene expression results in combination with the genome analysis suggest the possibility of long-range regulation of H2-T gene expression and/or important, but as yet unidentified nucleotide changes in the promoter or enhancer regions of the genes. Since the Mhc genomic region has diversified among mouse strains, it should be a useful model region for comparative analyses of the relationships between duplicated gene organization, evolution and the regulation of expression patterns.

Ultrastructural localization of prolactin in the rat anterior pituitary glands by preembedding peroxidase-labeled antibody method: observations in normal, castrated, or estrogen-stimulated specimen.

Ultrastructural localization of prolactin (PRL) was studied immunocytochemically (preembedding peroxidase-labeled antibody method) in a variety of pituitaries, including those from 1) normal, 2) castrated, and 3) castrated and estrogen-stimulated rats. In the normal rat, PRL was observed in cisternae of the rough endoplasmic reticulum (RER), perinuclear spaces, Golgi saccules, and secretory granules. In the castrated rats, PRL cells were rather atrophic and were filled with many small PRL-positive secretory granules. RER and Golgi saccules were rather inconspicuous and were almost devoid of PRL localization. The serum PRL level was markedly lowered. With estrogen stimulation after castration, the serum PRL level was markedly elevated and PRL cells showed a pronounced increase of PRL filled cisternae in the RER. From these observations, the role of secretory granules, Golgi apparatus, and RER in hormonal secretion was defined, and it was postulated that some peptide hormones would be secreted along two alternative pathways, i.e., either 1) a long (regulated) pathway or 2) a short (accelerated) pathway, in accordance with their secretory activities, which could be altered by various stimulations such as the use of estrogen.

Exact ultrastructural localization of glutathione peroxidase in normal rat hepatocytes: advantages of microwave fixation.

Glutathione peroxidase (GSH-PO), a highly soluble, selenium-dependent enzyme metabolizing lipid peroxides, is allegedly distributed in both the cytosol and mitochondria. With the pre-embedding method of immunoelectron microscopy for GSH-PO employing conventional immersion-fixation, the nuclei of rat hepatocytes stain positively, whereas mitochondria are negative. Such observations are inconsistent with the results of biochemical and immunoblot analyses using isolated subcellular fractions. In the present study, we employed the combination of microwave irradiation and fixation in 4% paraformaldehyde (PFA), with or without 0.1% glutaraldehyde (GA), to enhance the accuracy of ultrastructural localization of GSH-PO in rat liver. A small block of liver was irradiated by microwave for 10 sec in cold cacodylate-buffered 4% PFA containing 0.1% GA. After further immersion of the tissue in 4% PFA at 4 degrees C for 1-6 hr, the standard procedure for pre-embedding immunoelectron microscopy was employed. We observed partial inhibition of artifactual diffusion of cytosolic GSH-PO into the nuclei and consistent GSH-PO localization in mitochondria. Dual localization of this enzyme in the cytosol and mitochondria of normal rat hepatocytes was thus confirmed.

Relationship between oxidative stress in muscle tissue and weight-lifting-induced muscle damage

We examined whether oxidative stress-induced muscle damage occurs during weight-lifting exercise using the rat model. Male Wistar rats were subjected to a single exhaustive session of weight-lifting exercise, and dynamics of blood volume and hemoglobin levels in the exercising muscle were monitored by near-infrared spectroscopy. Total muscle damage was evaluated by the efflux of serum creatine kinase (CK) and uptake of [3H]thymidine. The production of reactive oxygen species (ROS) in the muscle was estimated by serial changes in total superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase (CAT) activities (established indirect markers). Immunohistochemical detection of GPX was also performed. A relatively anoxic state occurred repeatedly after every exercise set in exercising muscle following rapid blood reperfusion and was similar to an ischemia–reperfusion state. Serum CK and mitotic activity in the muscle consistently increased, and damaged muscle fibers that reacted positively to anti-GPX antibody were also observed after exercise. Serial changes in total SOD, GPX, and CAT activities were biphasic and exhibited peaks immediately and 24–72xa0h after exercise. The first increase was caused by a repeated ischemia–reperfusion-like state following weight-lifting exercise, and the second was dependent on the accumulation of infiltrated phagocytic cells at the damaged portions. These results suggest that ROS-induced muscle fiber damage occurred as a consequence of weight-lifting exercise.

Expression of Ptx1 in the adult rat pituitary glands and pituitary cell lines: hormone-secreting cells and folliculo-stellate cells.

Abstractu2002The pituitary homeobox1 gene (Ptx1) was initially identified as encoding a pituitary-restricted transcription factor for the proopiomelanocortin (POMC) gene. In order to elucidate the expression pattern of the Ptx1 protein, we investigated the localization of the protein in adult rat pituitary gland and in various pituitary cell lines. We produced an antibody specific for Ptx1 protein, and confirmed its specificity by Western blot analysis. Immunohistochemically, many nuclei in the anterior pituitary cells as well as in the intermediate cells were positive for Ptx1 staining with this specific antibody. Immunohistochemical double staining revealed the presence of Ptx1 not only in all types of hormone-secreting cells but also in some folliculo-stellate (FS) cells. Furthermore, the expression of Ptx1 mRNA was confirmed in various pituitary cell lines and in the FS cell line by using the reverse transcriptase-polymerase chain reaction (RT-PCR) method. Our studies indicated that Ptx1 may not only play a role as a basic transcriptional factor for production of various hormones, but may also play some important role(s) in FS cells. Possible synergistic actions with other factors remain to be investigated. The novel finding of Ptx1 in FS cells is of particular interest, and may suggest that FS cells and hormone-secreting cells are derived from a common cellular ancestor.

Immunoglobulins in cerebrospinal fluid: Enzyme-immunoassay

Using an enzyme-immunoassay (EIA) technique we established control values for IgA and IgM in cerebrospinal fluid (CSF). These values, together with IgG values determined by single radial immunodiffusion (SRID) technique, showed significant positive correlations with CSF total protein values. The CSF IgA and IgM levels were related to corresponding levels determined in serum. In addition, the values for all 3 immunoglobulin classes in CSF as well as CSF total protein values showed positive correlation with age of subjects, and IgG% and IgA% inreased with age. This new EIA procedure can be completed within 24 h and is sensitive enough for determining all 3 immunoglobulin classes using a small amount (100 microliter or less) of native CSF.