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Biomedical subjects

Hiroshi Imai

Publications and source records attributed to Hiroshi Imai.

At least 19 recordsLinked to original sources

HER2-specific T-cell immune responses in patients vaccinated with truncated HER2 protein complexed with nanogels of cholesteryl pullulan.

PURPOSE: We developed a complex of tumor antigen protein with a novel nanoparticle antigen delivery system of cholesteryl pullulan (CHP). To target HER2 antigen, we prepared truncated HER2 protein 1-146 (146HER2) complexed with CHP, the CHP-HER2 vaccine. We designed a clinical study to assess the safety of the vaccine and HER2-specific T-cell immune responses measured by the newly developed enzyme-linked immunospot assay with mRNA-transduced phytohemagglutinin-stimulated CD4(+) T cells in HLA-A2402-positive patients with therapy-refractory HER2-expressing cancers. EXPERIMENTAL DESIGN: Nine patients with various types of solid tumors were enrolled. Each patient was s.c. vaccinated biweekly with 300 microg of CHP-HER2 vaccine for three times followed by booster doses. HER2-specific T-cell responses were evaluated by enzyme-linked immunospot assay by targeting autologous phytohemagglutinin-stimulated CD4(+) T cells transduced with 146HER2-encoding mRNA to cover both identified peptides and unknown epitopes for MHC class I and class II that might exist in the sequence of the vaccine protein. RESULTS: CHP-HER2 vaccine was well tolerated; the only adverse effect was grade 1 transient skin reaction at the sites of vaccination. HER2-specific CD8(+) and/or CD4(+) T-cell immune responses were detected in five patients who received four to eight vaccinations, among whom both T-cell responses were detected in these patients. In four patients with CD8(+) T-cell responses, two patients reacted to previously identified HER2(63-71) peptide and the other two reacted only to 146HER2 mRNA-transduced cells. CONCLUSIONS: CHP-HER2 vaccine was safe and induced HER2-specific CD8(+) and/or CD4(+) T-cell immune responses.

Adult↗

Effects of synchronization of donor cell cycle on embryonic development and DNA synthesis in porcine nuclear transfer embryos.

The relationship between donor cell cycle and the developmental ability of somatic cell nuclear transfer (SCNT) embryos has not fully been elucidated. Donor cells that are usually prepared by serum starvation or confluent-cell culture for SCNT represent a heterogeneous population that includes mainly G0 phase cells, other cells in different phases of the cell cycle and apoptotic cells. In this study, we compared the developmental ability of porcine SCNT embryos reconstructed from G0 phase cells (G0-SCNT embryos) and strictly synchronized-G1 phase cells (G1-SCNT embryos), and examined the developmental rates and timing of first DNA synthesis. The G0 phase cells were synchronized by confluent culture, and the G1 phase cells were prepared from actively dividing M phase cells. The G1-SCNT embryos showed a significantly higher (P<0.05) developmental rate to the blastocyst stage per cleaved embryo (59%) than the G0-SCNT embryos (43%). Moreover, initiation of first DNA synthesis and cleavage occurred significantly earlier in the G1-SCNT embryos than in the G0-SCNT embryos. Delay of initiation of first DNA synthesis in the SCNT embryos by aphidicolin resulted in decreased developmental rates to the blastocyst stage without any effect on cleavage rates. Our data demonstrates that synchronized-G1 phase cells can be used as donor cells for SCNT embryos and that earlier initiation of first DNA synthesis may be important for subsequent development of SCNT embryos. The SCNT system using G1-synchronized cells, in terms of their highly uniform and viable cell states, can be useful for studying the reprogramming processes and embryonic development of SCNT embryos.

Animals↗

Identification and characterization of two novel classes of small RNAs in the mouse germline: retrotransposon-derived siRNAs in oocytes and germline small RNAs in testes.

Small RNAs ranging in size between 18 and 30 nucleotides (nt) are found in many organisms including yeasts, plants, and animals. Small RNAs are involved in the regulation of gene expression through translational repression, mRNA degradation, and chromatin modification. In mammals, microRNAs (miRNAs) are the only small RNAs that have been well characterized. Here, we have identified two novel classes of small RNAs in the mouse germline. One class consists of approximately 20- to 24-nt small interfering RNAs (siRNAs) from mouse oocytes, which are derived from retroelements including LINE, SINE, and LTR retrotransposons. Addition of retrotransposon-derived sequences to the 3' untranslated region (UTR) of a reporter mRNA destabilizes the mRNA significantly when injected into full-grown oocytes. These results suggest that retrotransposons are suppressed through the RNAi pathway in mouse oocytes. The other novel class of small RNAs is 26- to 30-nt germline small RNAs (gsRNAs) from testes. gsRNAs are expressed during spermatogenesis in a developmentally regulated manner, are mapped to the genome in clusters, and have strong strand bias. These features are reminiscent of Tetrahymena approximately 23- to 24-nt small RNAs and Caenorhabditis elegans X-cluster small RNAs. A conserved novel small RNA pathway may be present in diverse animals.

3' Untranslated Regions↗

Impact of lymph node micrometastasis in hilar bile duct carcinoma patients.

AIM: To immunohistochemically examine micrometastasis and VEGF-C expression in hilar bile duct carcinoma (HBDC) and to evaluate the clinical significance of the results. METHODS: A total of 361 regional lymph nodes from 25 patients with node-negative HBDC were immunostained with an antibody against cytokeratins 8 and 18 (CAM 5.2), and immunohistochemical staining of VEGF-C was performed in 34 primary resected tumors. RESULTS: Lymph node micrometastasis was detected in 6 (24%) of the 25 patients and 10 (2.8%) of the 361 lymph nodes. Patients with micrometastasis showed significantly poorer survival rates than those without (P = 0.025). VEGF-C expression was positive in 17 (50%) of 34 HBDC, and significantly correlated with lymph node metastasis (P = 0.042) and microscopic venous invasion (P = 0.035). CONCLUSIONS: It is suggested that immunohistochemically detected lymph node micrometastasis has an impact on the outcome of HBDC. VEGF-C expression is highly correlated with lymph node metastasis in HBDC and might therefore be a useful predictor.

Adult↗

Two-dimensional averaged images of the dynactin complex revealed by single particle analysis.

The dynactin complex interacts with dynein and numerous other proteins to provide for a wide range of subcellular transport functions. A detailed understanding of the structure and subunit organization of dynactin should yield new insights into its function. In the present study, we used single particle analysis to obtain a two-dimensional averaged image of dynactin isolated from chick embryo brains and visualized by negative stain electron microscopy (EM). Each individual image, consisting of the shoulder/sidearm and the rod, closely resembled the previously published quick-freeze deep-etch rotary-shadow electron micrographs. However, the averaged image revealed novel structural features that may have functional significance. The bulky shoulder complex has a triangular shape and is 13 nm wide and 8 nm high. The rod, with an overall length of 40 nm, consists of clearly defined lobes that are apparently grouped into three parts, the pointed-end complex, the middle segment, and the extra lobes at the barbed end. The pointed-end complex reveals the characteristic protrusions and clefts that were previously observed only in the isolated pointed-end complex. In the middle segment, the seven lobes are fitted to the helical symmetry of F-actin. A narrow but prominent gap separates the previously unidentified extra three lobes at the barbed end from the middle segment. The averaged image we obtained contrasts dramatically with the simple Arp1 polymer that was previously reported by single particle analysis of bovine brain dynactin. These apparent structural differences are probably due to the greater stability and integrity of the chick embryo brain dynactin preparation. We propose a new structural model for dynactin, based on our observations.

Actin Cytoskeleton↗

Oog1, an oocyte-specific protein, interacts with Ras and Ras-signaling proteins during early embryogenesis.

We previously identified an oocyte-specific gene, Oogenesin 1 (Oog1), that encodes 326 amino acids containing a leucine zipper structure and a leucine-rich repeat. In the present study, to identify the interacting proteins of Oog1, we performed a yeast two-hybrid screening using a GV-oocyte cDNA library and found that Ral guanine nucleotide dissociation stimulator (RalGDS) is the binding partner of Oog1. Coimmunoprecipitation assay confirmed the interaction between Oog1 and RalGDS proteins. Colocalization experiments provide the evidence that the nuclear localization of RalGDS depends on the expression of Oog1. Interestingly, RalGDS protein localized in the nucleus rather than the cytoplasm between late 1-cell and early 2-cell stages, the time when Oog1 localizes in the nucleus. We also examined the interaction between Oog1 and Ras by GST pull-down assay and revealed that Oog1 interacts with Ras in a GTP-dependent manner. These findings suggest a role of Oog1 as a Ras-binding protein.

Animals↗

A magnetically collimated jet from an evolved star.

Planetary nebulae often have asymmetric shapes, even though their progenitor stars were symmetric; this structure could be the result of collimated jets from the evolved stars before they enter the planetary nebula phase. Theoretical models have shown that magnetic fields could be the dominant source of jet-collimation in evolved stars, just as these fields are thought to collimate outflows in other astrophysical sources, such as active galactic nuclei and proto-stars. But hitherto there have been no direct observations of both the magnetic field direction and strength in any collimated jet. Here we report measurements of the polarization of water vapour masers that trace the precessing jet emanating from the asymptotic giant branch star W43A (at a distance of 2.6 kpc from the Sun), which is undergoing rapid evolution into a planetary nebula. The masers occur in two clusters at opposing tips of the jets, approximately 1,000 au from the star. We conclude from the data that the magnetic field is indeed collimating the jet.

Journal Article↗

Zygotically activated genes are suppressed in mouse nuclear transferred embryos.

Mammalian oocytes have the ability to confer totipotency to terminally differentiated somatic cell nuclei. Viable cloned animals have been produced by somatic cell nuclear transfer (NT) into oocytes in many mammalian species including mouse. However, the success rates of the production were quite low in all species. Many studies have measured differences in gene expression between NT and fertilized embryos in relatively advanced stages of development such as pre- and post-natal stages or the blastocyst stage. In the present study, we compared gene expression patterns using differential display RT-PCR (DDRT-PCR) between the NT and IVF embryos at the 2-cell stage to detect some abnormalities affecting later development of NT embryos. Aberrant gene expression was detected in NT embryos compared with IVF embryos, and MuERV-L and Dnaja2 genes were down-regulated and Inpp5b and Chst12 genes were up-regulated in the NT embryos. Further analysis showed that the expression of zygotically activated genes such as Interferon-gamma, Dub-1, Spz1, DD2106 (unknown gene), and DD2111 (unknown gene) were suppressed in NT embryos, suggesting that the cellular process involved in the nuclear reprogramming of somatic nucleus is not appropriately regulated.

Animals↗

Properties of Orange-Pupa-Inducing Factor (OPIF) in the swallowtail butterfly, Papilio xuthus L.

Diapause pupae of the swallowtail butterfly Papilio xuthus L. exhibit diapause-green, orange and brownish-orange color polymorphism. Development of orange pupae involves a neuroendocrine factor inducing orange pupa (Orange-Pupa-Inducing Factor, OPIF), which is secreted from the head-thoracic region during late pharate pupal stages, in particular from the ganglia of short-day animals located posteriorly from the second thoracic ganglion2 (TG2). This report describes certain properties of OPIF using bioassays involving ligated abdomens of short-day pharate pupae. Localization of OPIF in the central nervous system of short-day larvae indicated that it was present predominantly in TG2, thoracic ganglion3 (TG3) and abdominal ganglion1 (AG1) complexes. OPIF activity in TG(2,3)-AG1 complexes was over two times higher than in the more posteriorely located ganglia. The developmental profile of OPIF in last instar short-day larvae revealed that OPIF activity in larval ganglia posterior to TG2 became gradually higher as larval growth proceeded, suggesting that OPIF might be accumulated in TG(2,3)-AG(1-7) complexes as larvae prepare for pupal molting. Furthermore, ligated abdomens of short-day larvae developed into pupae of an orange type when a 2% NaCl extract containing OPIF prepared from TG(2,3)-AG(1-7) complexes of long-day larvae was injected into ligated abdomens of short-day pharate pupae, indicating that OPIF is also present in long-day larvae. Additionally, a biochemical investigation using gel filtration chromatography showed that the molecular weight of OPIF was about 10 kDa.

Animals↗

Neurochondrin negatively regulates CaMKII phosphorylation, and nervous system-specific gene disruption results in epileptic seizure.

Neurochondrin is a novel cytoplasmic protein and possibly involved in neurite outgrowth, chondrocyte differentiation, and bone metabolism. Our previous trial in disclosing its role by the loss of function in mice failed because of the lethality in utero. In this study, we eliminated the neurochondrin gene expression preferentially in the nervous system by the conditional knockout strategy. Our results showed that neurochondrin is a negative regulator of Ca(2+)/calmodulin-dependent protein kinase II phosphorylation and essential for the spatial learning process but not for the differentiation or neurite outgrowth of the neuron. In addition, the nervous system-specific homozygous gene disruption resulted in epileptic seizure.

Animals↗

Stage-specific expression of microRNAs during Xenopus development.

MicroRNAs (miRNAs) repress target genes at the post-transcriptional level and play important roles in development and cell lineage decision. However, in vertebrates, both the targets of miRNAs and their expression profile during development are poorly understood. Here, we report the detailed expression profiles of miRNAs from oocyte stage to tadpole stage in Xenopus laevis. As development proceeds, a variety of miRNAs start to be expressed. Most miRNAs emerged at a specific stage and were continuously expressed until the tadpole stage. In addition, we identified a novel miRNA that was expressed only at specific stages of development and that is likely to have roles in midblastula transition.

Animals↗

Developmental competence of somatic cell nuclear transfer embryos reconstructed from oocytes matured in vitro with follicle shells in miniature pig.

The developmental competence of domestic pig oocytes that were transferred to somatic cell nuclei of miniature pig was examined. A co-culture system of oocytes with follicle shells was used for the maturation of domestic pig oocytes in vitro. Co-cultured oocytes progressed to the metaphase II stage of meiosis more quickly and more synchronously than non co-cultured oocytes. Oocytes were enucleated and fused with fibroblast cells of Potbelly miniature pig at 48 h of maturation. The blastocyst formation rate of nuclear transfer (NT) embryos using cocultured oocytes (24%) was significantly higher (p < 0.05) than that of non-co-cultured oocytes (13%). Cleaved embryos at 48 h after nuclear transfer using co-cultured oocytes were transferred to the oviducts of 14 Göttingen miniature pigs and four Meishan pigs. Estrus of all Göttingens returned at around 20-31 days of pregnancy. Two of the four Meishans became pregnant. Three and two cloned piglets were born after modest number of embryo transfer (15 and 29 embryos transferred), respectively. These results indicated that oocytes co-cultured with follicle shells have a high developmental competence after nuclear transfer and result in full-term development after embryo transfer.

Animals↗

The roles of vitamin A for cytoplasmic maturation of bovine oocytes.

Vitamin A is one of the micronutrients which have been implicated in cattle reproduction. In cattle, ingested vitamin A, mainly as beta-carotene (BC) from forages and retinol ester from formula feed, is metabolized and transported to the oocytes and cumulus-granulosa cells in ovarian follicles through binding to various interacting molecules. The active form of vitamin A, retinoic acid (RA), functions as a regulator of gene expression in these targets. Early research showed the positive effects of vitamin A supplementation on bovine fertility in artificial insemination, and several studies on effects of vitamin A metabolites used in other artificial reproductive techniques (ART), including superovulation, ovum pick up, and in vitro maturation culture have provided evidence for the specific roles of vitamin A in oocyte cytoplasmic maturation (acquisition of developmental competence of oocytes during their meiotic maturation period for the embryonic development after fertilization). BC may enhance cytoplasmic maturation by its antioxidant properties which cannot be replaced by RA. Furthermore, RA may promote cytoplasmic maturation of bovine oocytes via its modulatory effects on the gene expression of gonadotrophin receptors, midkine, cyclooxygenase-2, and nitric oxide synthase in cumulus-granulosa cells.

Animals↗

[Histopathological features of prostate cancer].

The diagnosis of prostatic cancer present in a limited amount within needle biopsy tissue, is often challenging. The most common mimickers giving rise to false-positive cancer diagnosis are atypical adenomatous hyperplasia, prostatic intraepithelial neoplasia, atrophy and post-atrophic hyperplasia. Various diagnostic criteria including assessment of basal cells should be used for diagnosis of limited carcinoma. Immunohistochemical staining for both basal cells, such as 34betaE12 and p63, and AMACR, which label the cytoplasm of approximately 80% of prostatic adenocarcinoma, may be a useful adjunct in the diagnosis of limited prostatic cancer. However there are problems with both sensitivity and specificity. When the glands lacking sufficient criteria to establish a definitive carcinoma is present, we use the term 'atypical small acinar proliferation'.

Adenocarcinoma↗

Intravascular large B-cell lymphoma presenting with mass lesions in the central nervous system: a report of five cases.

We have encountered five cases of intravascular large B-cell lymphoma (IVL) presenting with central nervous system (CNS) mass lesions during their clinical course. The age of the patients ranged from 50 to 74 years and three patients were male. All of these cases histopathologically showed typical intravascular localization of the neoplastic cells in the initial biopsy specimens obtained from sites other than the CNS. Despite multiagent chemotherapy, patients suffered from single or multiple CNS mass lesions 5-44 months after the initial diagnosis of IVL, except for one case in which IVL and the CNS mass lesion were diagnosed at the same time. The subsequent biopsy and autopsy specimens obtained from the CNS mass lesions revealed diffuse infiltration of the tumor cells with perivascular spreading, but minimal or no intravascular components. Immunohistochemical analysis of intravascular tumor cells and CNS mass lesions revealed expression of CD20, CD79a, bcl-2 and negative for CD3e and Epstein-Barr virus encoded RNA. The overall features of the CNS mass lesions were very similar to or indistinguishable from those of the primary CNS lymphomas. This implies that CNS mass lesions in the IVL cases can be correctly diagnosed only by careful attention to clinical and pathological findings. Moreover, there is the possibility that some cases previously diagnosed as primary CNS lymphomas may have include IVL cases. Further investigation is needed to explore this unusual phenomenon.

Aged↗