尾之内 高慶

Regulation of ion and water microcirculation within the lens is tightly controlled through aquaporin channels and connexin junctions. However, cataracts can occur when the lens becomes cloudy. Various factors can induce cataracts, including diabetes which is a well-known cause. The most common phenotype of diabetic cataracts is a cortical and/or posterior subcapsular opacity. In addition to the three main types and two subtypes of cataracts, a vacuole formation is frequently observed; however, their origin remains unclear. In this study, we focused on the aquaporins and connexins involved in diabetes-induced cataracts and vacuoles in Nile grass type II diabetes. The results showed that the expression of aquaporin 0 and aquaporin 5 increased, and that of connexin 43 decreased in diabetic rat lenses. Additionally, aquaporin 0 and 5 were strongly localized in peripheral of vacuoles, suggesting that aquaporins are involved in vacuoles formation. Transillumination photography revealed large vacuoles at the tip of the Y-suture in the anterior capsule of the diabetic lens, and several small vacuoles were observed in the posterior capsule. Within the vacuoles, cytoplasmic degradation and aggregation of fibrous material were observed. Our findings suggest that aquaporins are potential candidate proteins for preventing vacuole formation.

EBV infections cause nucleolar enlargement via the induction of IMPDH2, which are essential for B cell growth transformation by EBV. Although the significance of IMPDH2 induction and nuclear hypertrophy in the tumorigenesis of glioblastoma has been reported, EBV infection brings about the change quickly by using its transcriptional cofactor, EBNA2, and MYC. Moreover, we present here, for the novel, basic evidence that an IMPDH2 inhibitor, namely, MPA or MMF, can be used for EBV-positive posttransplant lymphoproliferative disorder (PTLD).

Huntingtin-associated protein 1 (HAP1) is abundantly expressed in the neurons of the central nervous system and forms unique intracytoplasmic inclusions of unknown function called "stigmoid bodies" (STBs). Transmission electron microscopy has revealed that the STBs are aggregates of granules containing cavities with a diameter of 0.5-3 µm. Small STBs fuse to form larger STBs, the size of which is said to vary depending on the developmental growth stage and brain region. Light microscopy can only reveal that these STBs have similar circular shapes, due to its limited resolution. Therefore, light microscopy is only fit for the study of the STB distribution and quantitative changes. We, herein, suggest the adoption of correlative light and electron microscopy, that combines confocal laser scanning microscopy and scanning electron microscopy, as the method allowing us to identify the HAP1-positive STBs in formalin-fixed paraffin-embedded (FFPE) sections. This approach allows us to study the three-dimensional morphology of immunolabeled objects in histopathological specimens. The STBs in FFPE sections of murine hypothalami reflected the transmission electron microscopic images of Epon-embedded STBs, although we were not able to observe any organelle covering the STBs of the FFPE sections. Furthermore, we were able to reconstruct the three-dimensional structure of the STB and we identified it to be of spherical form, covered with mitochondria and rough endoplasmic reticulum, and bearing a cluster of cavities in the center. In the future, we might gain new insights by comparing the 3D structure of the STB between different neurons and under a variety of conditions.

Neutrophil extracellular trap (NET) formation, an innate immune system response, is associated with thrombogenesis and vascular endothelial injury. Circulatory disorders due to microvascular thrombogenesis are one of the principal causes of organ damage. NET formation in organs contributes to the exacerbation of sepsis, which is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection. We have previously reported that recombinant human soluble thrombomodulin (rTM) reduces lipopolysaccharide (LPS)-induced NET formation in vitro. Here, we aimed to show that thrombomodulin (TM)-mediated suppression of NET formation protects against organ damage in sepsis. Mice were injected intraperitoneally (i.p.) with 10 mg/kg LPS. rTM (6 mg/kg/day) or saline was administered i.p. 1 h after LPS injection. In the LPS-induced murine septic shock model, extracellular histones, which are components of NETs, were observed in the liver and lungs. In addition, the serum cytokine (interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), macrophage chemotactic protein-1 (MCP-1), and interleukin-10 (IL-10)) levels were increased. The administration of rTM in this model prevented NET formation in the organs and suppressed the increase in the levels of all cytokines except IL-1β. Furthermore, the survival rate improved. We provide a novel role of TM in treating inflammation and NETs in organs during sepsis.

In the present study, we examined morphology and function of hippocampus in the APC1638T/1638T mouse. Expression levels of the APC mRNA and protein were both identical in the hippocampus of the APC+/+ and APC1638T/1638T mice. The dentate gyrus of the APC1638T/1638T hippocampus was thicker, and has more densely-populated granule cells in the APC1638T/1638T mouse hippocampus. Immunoelectron microscopy revealed co-localization of APC with alpha-amino-3- hydroxy-5-methyl- isoxazole-4-propionate receptor (AMPA-R) and with PSD-95 at post-synapse in the APC+/+ hippocampus, while APC1638T was co-localized with neither AMPA-R nor PSD-95 in the APC1638T/1638T hippocampus. By immunoprecipitation assay, full-length APC expressed in the APC+/+ mouse was co-immunoprecipitated with AMPA-R and PSD-95. In contrast, APC1638T expressed in the APC1638T/1638T mouse was not co-immunoprecipitated with AMPA-R and PSD-95. In the hippocampal CA1 region of the APC1638T/1638T mouse, c-Fos expression after electric foot shock was decreased compared with the APC+/+ mouse. The present study showed some abnormalities on morphology of the hippocampus caused by a truncated APC (APC1638T). Also, our findings suggest that failure in APC binding to AMPA-R and PSD-95 may bring about less activities of hippocampal neurons in the APC1638T/1638T mouse.

Dendritic cell-based immunotherapy, which uses a patient's own immune cells, can be used for cancer treatment and allergy control, such as autoimmune disease and rejection associated with transplantation. However, these treatments create a burden on patients due to repeated blood collection. We used cell biological analysis of monocytes with few mutations obtained from minimal blood collection for genome recombination. Next, we established human peripheral blood monocyte-derived induced pluripotent stem cells (iPSCs) using a commercial vector and standard culture method. We found that when established iPSCs were induced to differentiate, monocytes showed phagocytic properties and expressed CD14 and CX3CR1. Further, the generated dendritic cells (DCs) expressed CCL17 and highly expressed HLA-DR following the addition of the mite antigen. Taken together, these data show that monocyte-derived iPS cells can be used to differentiate into monocytes and DCs. In addition, the use of these cells can be applied to the pathological analysis of dendritic cell therapy and monocyte diseases.

Introduction: Cribriform-morular variant (CMV) is a rare variant of papillary thyroid carcinoma. It frequently occurs in association with familial adenomatous polyposis (FAP), although some cases are sporadic. Herein, we report a case of CMV and analyse morule cytohistology. Case Report: The patient was a 47-year-old woman with no familial history of FAP. A 3.0-cm unifocal mass was identified in the left thyroidal lobe. Fine-needle aspiration cytology revealed papillary clusters of atypical cells with nuclear grooves, which was suspected to be conventional papillary thyroid carcinoma. Histologically, the tumour comprised a papillary and cribriform growth of atypical cells with cytoplasmic accumulation and nuclear translocation of b-catenin. In addition, frequent morule formation was identified. Discussion: In this case, we performed morule analysis through correlative light and electron microscopy (CLEM), and revealed its ultrastructure. Although CMV is a rare form of thyroid carcinoma, it should be considered along with its distinct clinicopathological characteristics.

Exosomes, a type of small extracellular vesicles (sEVs), derived from multivesicular bodies (MVBs), mediate cell-to-cell communication by transporting proteins, mRNAs, and miRNAs. However, the molecular mechanism by which proteins are sorted to sEVs is not fully understood. Here, we report that ubiquitin-like 3 (UBL3)/membrane-anchored Ub-fold protein (MUB) acts as a posttranslational modification (PTM) factor that regulates protein sorting to sEVs. We find that UBL3 modification is indispensable for sorting of UBL3 to MVBs and sEVs. We also observe a 60% reduction of total protein levels in sEVs purified from Ubl3-knockout mice compared with those from wild-type mice. By performing proteomics analysis, we find 1241 UBL3-interacting proteins, including Ras. We also show that UBL3 directly modifies Ras and oncogenic RasG12V mutant, and that UBL3 expression enhances sorting of RasG12V to sEVs via UBL3 modification. Collectively, these results indicate that PTM by UBL3 influences the sorting of proteins to sEVs.

The adenomatous polyposis coli (APC) is a multifunctional protein as well as a tumor suppressor. To determine the functions of the C-terminal domain of APC, we explored APC 1638T/1638T (APC1638T) mice that express a truncated APC lacking the C-terminal domain. The APC1638T mice were tumor free and exhibited growth retardation. In the present study, we compared small intestinal crypt-villus cells homeostasis in APC +/+ (WT) mice and APC1638T mice. The body weight of APC1638T mice was significantly smaller than that of WT mice at all ages. The length of small intestine of APC1638T mice was significantly shorter than that of WT mice. The crypt-villus axis was significantly elongated, and the number of intestinal epithelial cells also increased in APC1638T mice compared with those in WT mice. However, the number of intestinal epithelial cells per 100 µm of villi was not different between WT and APC1638T mice. Migration and proliferation of intestinal epithelial cells in APC1638T mice were faster than that in WT mice. The population of Goblet cells, Paneth cells, and enteroendocrine cells was significantly altered in APC1638T mice. These results indicate that C-terminal domain of APC has a role in the regulation of intestinal epithelium homeostasis.

Neutrophil extracellular traps (NETs) released from dead neutrophils at the site of inflammation represent webs of neutrophilic DNA stretches dotted with granule-derived antimicrobial proteins, including lactoferrin, and play important roles in innate immunity against microbial infection. We have shown the coexistence of NETs and fibrin meshwork in varied fibrinopurulent inflammatory lesions at both light and electron microscopic levels. In the present study, correlative light and electron microscopy (CLEM) employing confocal laser scanning microscopy and scanning electron microscopy was performed to bridge light and electron microscopic images of NETs and fibrin fibrils in formalin-fixed, paraffin-embedded, autopsied lung sections of legionnaire's pneumonia. Lactoferrin immunoreactivity and 4'-6-diamidino-2-phenylindole (DAPI) reactivity were used as markers of NETs, and fibrin was probed by fibrinogen gamma chain. Of note is that NETs light microscopically represented as lactoferrin and DAPI-colocalized dots, 2.5 μm in diameter. CLEM gave super-resolution images of NETs and fibrin fibrils: "Dotted" NETs were ultrastructurally composed of fine filaments and masses of 58 nm-sized globular materials. A fibrin fibril consisted of clusters of smooth-surfaced filaments. NETs filaments (26 nm in diameter) were significantly thinner than fibrin filaments (295 nm in diameter). Of note is that CLEM was applicable to formalin-fixed, paraffin-embedded sections of autopsy material.

Neutrophil extracellular traps (NETs) are extracellular fibrillary structures composed of degraded chromatin and granules of neutrophil origin. In fibrinopurulent inflammation such as pneumonia and abscess, deposition of fibrillar eosinophilic material is a common histopathological finding under hematoxylin-eosin staining. Expectedly, not only fibrin fibrils but also NETs consist of the fibrillar material. The aim of the present study is to analyze immunohistochemically how NETs are involved in the inflammatory process. Archival formalin-fixed, paraffin-embedded sections accompanying marked neutrophilic infiltration were the target of analysis. Neutrophil-associated substances (citrullinated histone H3, lactoferrin, myeloperoxidase and neutrophil elastase) were evaluated as NETs markers, while fibrinogen gamma chain was employed as a fibrin marker. Light microscopically, the fibrils were categorized into three types: thin, thick and clustered thick. Lactoferrin represented a good and stable NETs marker. Thin fibrils belonged to NETs. Thick fibrils are composed of either mixed NETs and fibrin or fibrin alone. Clustered thick fibrils were solely composed of fibrin. Neutrophils were entrapped within the fibrilllar meshwork of the thin and thick types. Apoptotic cells immunoreactive to cleaved caspase 3 and cleaved actin were dispersed in the NETs. In conclusion, NETs and fibrin meshwork were consistently recognizable by immunostaining for lactoferrin and fibrinogen gamma chain.

Neutrophil extracellular traps (NETs) represent an extracellular, spider's web-like structure resulting from cell death of neutrophils. NETs play an important role in innate immunity against microbial infection, but their roles in human pathological processes remain largely unknown. NETs and fibrin meshwork both showing fibrillar structures are observed at the site of fibrinopurulent inflammation, as described in our sister paper [Acta Histochem. Cytochem. 49; 109-116, 2016]. In the present study, immunoelectron microscopic study was performed for visualizing NETs and fibrin fibrils (thick fibrils in our tongue) in formalin-fixed, paraffin-embedded sections of autopsied lung tissue of legionnaire's pneumonia. Lactoferrin and fibrinogen gamma chain were utilized as markers of NETs and fibrin, respectively. Analysis of immuno-scanning electron microscopy indicated that NETs constructed thin fibrils and granular materials were attached onto the NETs fibrils. The smooth-surfaced fibrin fibrils were much thicker than the NETs fibrils. Pre-embedding immunoelectron microscopy demonstrated that lactoferrin immunoreactivities were visible as dots on the fibrils, whereas fibrinogen gamma chain immunoreactivities were homogeneously observed throughout the fibrils. Usefulness of immunoelectron microscopic analysis of NETs and fibrin fibrils should be emphasized.

In chronic inflammatory lesions of autoimmune and infectious diseases, plasma cells are frequently observed. Antigens recognized by antibodies produced by the plasma cells mostly remain unclear. A new technique identifying these corresponding antigens may give us a breakthrough for understanding the disease from a pathophysiological viewpoint, simply because the immunocytes are seen within the lesion. We have developed an enzyme-labeled antigen method for microscopic identification of the antigen recognized by specific antibodies locally produced in plasma cells in inflammatory lesions. Firstly, target biotinylated antigens were constructed by the wheat germ cell-free protein synthesis system or through chemical biotinylation. Next, proteins reactive to antibodies in tissue extracts were screened and antibody titers were evaluated by the AlphaScreen method. Finally, with the enzyme-labeled antigen method using the biotinylated antigens as probes, plasma cells producing specific antibodies were microscopically localized in fixed frozen sections. Our novel approach visualized tissue plasma cells that produced 1) autoantibodies in rheumatoid arthritis, 2) antibodies against major antigens of Porphyromonas gingivalis in periodontitis or radicular cyst, and 3) antibodies against a carbohydrate antigen, Strep A, of Streptococcus pyogenes in recurrent tonsillitis. Evaluation of local specific antibody responses expectedly contributes to clarifying previously unknown processes in inflammatory disorders.

Ixodid ticks transmit several important viral pathogens. We isolated a new virus (Tofla virus: TFLV) from Heamaphysalis flava and Heamaphysalis formsensis in Japan. The full-genome sequences revealed that TFLV belonged to the genus Nairovirus, family Bunyaviridae. Phylogenetic analyses and neutralization tests suggested that TFLV is closely related to the Hazara virus and that it is classified into the Crimean-Congo hemorrhagic fever group. TFLV caused lethal infection in IFNAR KO mice. The TFLV-infected mice exhibited a gastrointestinal disorder, and positron emission tomography-computed tomography images showed a significant uptake of (18)F-fluorodeoxyglucose in the intestinal tract. TFLV was able to infect and propagate in cultured cells of African green monkey-derived Vero E6 cells and human-derived SK-N-SH, T98-G and HEK-293 cells. Although TFLV infections in humans and animals are currently unknown, our findings may provide clues to understand the potential infectivity and to develop of pre-emptive countermeasures against this new tick-borne Nairovirus.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging disease that causes fever, enteritis, thrombocytopenia, and leucopenia and can be fatal in up to 30% of cases. However, the mechanism of severe disease is not fully understood. Molecular imaging approaches, such as positron-emission tomography (PET), are functional in vivo imaging techniques that provide real-time dynamics of disease progression, assessments of pharmacokinetics, and diagnoses for disease progression. Molecular imaging also potentially provides useful approaches to explore the pathogenesis of viral infections. Thus, the purpose of this study was to image the pathological features of SFTSV infection in vivo by PET imaging. In a mouse model, we showed that 18F-FDG accumulations clearly identified the intestinal tract site as a pathological site. We also demonstrated that 18F-FDG PET imaging can assess disease progression and response to antiserum therapy within the same individual. This is the first report demonstrating a molecular imaging strategy for SFTSV infection. Our results provide potentially useful information for preclinical studies such as the elucidation of the mechanism of SFTSV infection in vivo and the assessment of drugs for SFTS treatment.

Particulated structures of microbes are relatively resistant to paraffin embedding. Nuclear/cytoplasmic inclusions in paraffin-embedded H&amp E preparation or ethanol-fixed Papanicolaou-stained cytologic specimens can be targeted for EM observation to demonstrate the microbes within the cell [1-3]. The antigens of Chlamydia trachomatis, E. coli, cytomegalovirus and human papillomavirus have been localized in paraffin sections by applying pre-embedding immuno-EM [2, 4, 5]. Although the fine morphologic preservation is not good enough, this approach is useful to confirm the presence of microbes in the lesions and the specificity of the antibodies. The viral genomes are also identifiable in routine samples by in situ hybridization at the EM level [1, 2, 4, 5]. NETs represent an extracellular, spider’s web-like structure resulting from cell death of neutrophils to trap and kill microbes. NETs often co-exist with fibrin meshwork at the site of fibrinopurulent inflammation. Immuno-EM study and correlative light and electron microscopy (CLEM), employing confocal laser scanning microscopy and scanning EM, were performed for visualizing NETs and fibrin fibrils in autopsied legionnaire’s pneumonia [6-8]. Lactoferrin and fibrinogen gamma chain marked NETs and fibrin, respectively. Pre-embedding immuno-EM, immuno-scanning EM and CLEM indicated that NETs constructed thin fibrils with granular materials attached. Smooth-surfaced fibrin fibrils were much thicker than the NETs fibrils, and each fibril consisted of a cluster of fine filaments.

We performed pre-embedding electron microscopic study for visualizing the antigen and genome of severe fever with thrombocytopenia syndrome (SFTS) virus in the cytoplasm of macrophages of the human splenic red pulp, both requesting preheating treatment of sections. To pursue this, coated glass slides with unique characteristics are needed. Namely, during staining they must prevent detaching off sections, but after staining the sections must be transferred to epoxy resin. Aminopropyltriexoxysilane-coated glass slides, widely used for immunostaining, were resistant to transfer to epoxy resin. In contrast, coated glass slides designated as Thinlayer Advanced Cytology Assay System (TACAS) were suitable for this purpose. The technique is also applicable to the coated glass slide-requiring cytology practice, in which immunocytochemical evaluation is needed after cell transfer to another glass slide.

Streptococcus pyogenes is the main causative pathogen of recurrent tonsillitis. Histologically, lesions of recurrent tonsillitis contain numerous plasma cells. Strep A is an antigenic carbohydrate molecule on the cell wall of S. pyogenes. As expected, plasma cells in subjects with recurrent tonsillitis secrete antibodies against Strep A. The enzyme-labeled antigen method is a novel histochemical technique that visualizes specific antibody-producing cells in tissue sections by employing a biotin-labeled antigen as a probe. The purpose of the present study was to visualize plasma cells producing antibodies reactive with Strep A in recurrent tonsillitis. Firstly, the lymph nodes of rats immunized with boiled S. pyogenes were paraformaldehyde-fixed and specific plasma cells localized in frozen sections with biotinylated Strep A. Secondly, an enzyme-labeled antigen method was used on human tonsil surgically removed from 12 patients with recurrent tonsillitis. S. pyogenes genomes were PCR-detected in all 12 specimens. The emm genotypes belonged to emm12 in nine specimens and emm1 in three. Plasma cells producing anti-Strep A antibodies were demonstrated in prefixed frozen sections of rat lymph nodes, 8/12 human specimens from patients with recurrent tonsillitis but not in two control tonsils. In human tonsils, Strep A-reactive plasma cells were observed within the reticular squamous mucosa and just below the mucosa, and the specific antibodies belonged to either IgA or IgG classes. Our technique is effective in visualizing immunocytes producing specific antibodies against the bacterial carbohydrate antigen, and is thus a novel histochemical tool for analyzing immune reactions in infectious disorders.

We established a new "ecological" Grocott stain for demonstrating fungi, based upon a 4R principle of refusal, reduction, reuse, and recycle of waste management. Conventional Grocott stain employs environmentally harsh 5% chromic acid for oxidization. Initially, we succeeded in reducing the concentration of chromic acid from 5% to 1% by incubating the solution at 60°C and using five-fold diluted chromic acid solution at which point it was reusable. Eventually, we reached the refusal level where 1% periodic acid oxidization was efficient enough, when combined with preheating of sections in the electric jar, microwave oven, or pressure pan. For convenience sake, we recommend pressure pan heating in tap water for 10 min. Stainability of fungi in candidiasis and aspergillosis was comparable with conventional Grocott stain, while Mucor hyphae showed enhanced staining. The modified sequence was further applicable to detecting a variety of mycotic pathogens in paraffin sections. Our environmentally-friendly Grocott stain also has the advantage of avoiding risk of human exposure to hexavalent chromium solution in the histopathology laboratory. The simple stain sequence is can be easily applied worldwide.

BACKGROUND: Loss of adenomatous polyposis coli (APC) gene function results in constitutive activation of the canonical Wnt pathway and represents the main initiating and rate-limiting event in colorectal tumorigenesis. APC is likely to participate in a wide spectrum of biological functions via its different functional domains and is abundantly expressed in the brain as well as in peripheral tissues. However, the neuronal function of APC is poorly understood. To investigate the functional role of Apc in the central nervous system, we analyzed the neurological phenotypes of Apc1638T/1638T mice, which carry a targeted deletion of the 3' terminal third of Apc that does not affect Wnt signaling. RESULTS: A series of behavioral tests revealed a working memory deficit, increased locomotor activity, reduced anxiety-related behavior, and mildly decreased social interaction in Apc1638T/1638T mice. Apc1638T/1638T mice showed abnormal morphology of the dendritic spines and impaired long-term potentiation of synaptic transmission in the hippocampal CA1 region. Moreover, Apc1638T/1638T mice showed abnormal dopamine and serotonin distribution in the brain. Some of these behavioral and neuronal phenotypes are related to symptoms and endophenotypes of schizophrenia. CONCLUSIONS: Our results demonstrate that the C-terminus of the Apc tumor suppressor plays a critical role in cognitive and neuropsychiatric functioning. This finding suggests a potential functional link between the C-terminus of APC and pathologies of the central nervous system.

<italic>Background</italic>.<italic>In situ</italic>hybridization (ISH) with high sensitivity has been requested to demonstrate hepatitis C virus (HCV) RNA in formalin-fixed, paraffin-embedded (FFPE) sections of the liver.<italic>Methods</italic>. ISH employing a locked-nucleic-acid- (LNA-)modified oligonucleotide probe and biotin-free catalyzed signal amplification system (CSAII) was applied to HCV-RNA detection in the liver tissue. Nested reverse-transcription polymerase chain reaction (RT-PCR) was performed for HCV genotyping using total RNA extracted from FFPE sections. The target tissues included FFPE tissue sections of humanized livers in HCV-infected chimeric mice (HCV genotypes 1a, 1b, and 2a and noninfected) and of needle-biopsied livers from HCV-infected patients.<italic>Results</italic>. HCV-RNA was demonstrated with the ISH technique in HCV-infected liver tissues from both chimeric mice and 9 (82%) of 11 patients with HCV infection. The HCV signals were sensitive to RNase. Nested RT-PCR confirmed the genotype in 8 (73%) of 11 livers (type 1b: 6 lesions and type 2a: 2 lesions). HCV-RNA was not identified in chronic hepatitis B lesions, fatty liver, autoimmune hepatitis, and hepatocellular carcinoma.<italic>Conclusion</italic>. ISH using the LNA-modified oligonucleotide probe and CSAII was applicable to detecting HCV-RNA in routinely prepared FFPE liver specimens.

Adenomatous polyposis coli protein (APC) is highly expressed in the nervous tissue, but its function there is not yet known. We previously found that the microtubule-bundling activity of APC is stimulated by its interaction with PSD-95, a neuronal scaffolding protein, in cultured COS-7 cells. In the present study, we investigated the distribution and localization of the intrinsic APC and PSD-95 in both cultured rat hippocampal neurons and in the rat cerebellum by immunofluorescence and immunoelectron microscopy. In cultured neurons, most of the PSD-95 immunofluorescence puncta were colocalized with APC, and the APC and PSD-95 immunogolds were colocalized in the nerve fibers as well as in the postsynaptic site, but not in the presynaptic site. In the molecular layer of the rat cerebellum, colocalization of APC and PSD-95 was also detected in the nerve fibers and the postsynaptic site, but not in the presynaptic site. Based on these results, we conclude that APC and PSD-95 colocalize and bind to form a protein complex in nerve fibers as well as in postsynaptic sites in matured neurons, suggesting the involvement of the APC/PSD-95 complex in the microtubule functions within the nerve fibers and the synapse functions at the postsynaptic site.

Adenomatous polyposis coli (Apc) is a multifunctional protein as well as a tumor suppressor. To determine the functions of the C-terminal domain of Apc, we examined Apc(1638T/1638T) mice that express a truncated Apc lacking the C-terminal domain. The Apc(1638T/1638T) mice were tumor free and exhibited growth retardation. We recently reported abnormalities in thyroid morphology and functions of Apc(1638T/1638T) mice, although the mechanisms underlying these abnormalities are not known. In the present study, we further compared thyroid gland morphology in Apc(1638T/1638T) and Apc(+/+) mice. The diameters of thyroid follicles in the left and right lobes of the same thyroid gland of Apc(1638T/1638T) mice were significantly different whereas the Apc(+/+) mice showed no significant differences in thyroid follicle diameter between these lobes. To assess the secretory activities of thyroid follicular cells, we performed double-immunostaining of thyroglobulin, a major secretory protein of these cells, and the rough endoplasmic reticulum (rER) marker calreticulin. In the Apc(1638T/1638T) follicular epithelial cells, thyroglobulin was mostly colocalized with calreticulin whereas in the Apc(+/+) follicular epithelial cells, a significant amount of the cytoplasmic thyroglobulin did not colocalize with calreticulin. In addition, in thyroid-stimulating hormone (TSH)-treated Apc(1638T/1638T) mice, electron microscopic analysis indicated less frequent pseudopod formation at the apical surface of the thyroid follicular cells than in Apc(+/+) mice, indicating that reuptake of colloid droplets containing iodized thyroglobulin is less active. These results imply defects in intracellular thyroglobulin transport and in pseudopod formation in the follicular epithelial cells of Apc(1638T/1638T) mice and suggest suppressed secretory activities of these cells.

Adenomatous polyposis coli (APC) is a multifunctional protein as well as a tumor suppressor. To determine the functions of the C-terminal domain of Apc, we have investigated Apc ( 1638T/1638T ) mice, which express a truncated Apc that lacks the C-terminal domain. Apc ( 1638T/1638T ) mice are tumor free and exhibit growth retardation. In the present study, we analyzed the morphology and functions of the thyroid gland in Apc ( 1638T/1638T ) mice. There was no significant difference in the basal concentration of serum thyroid hormones between Apc ( 1638T/1638T ) and Apc (+/+) mice. Thyroid follicle size was significantly larger in Apc ( 1638T/1638T ) mice than in Apc (+/+) mice. The extent of serum T4 elevation following exogenous thyroid-stimulating hormone (TSH) injection was lower in Apc ( 1638T/1638T ) mice than in Apc (+/+) mice. TSH also induced a greater reduction in thyroid follicle size in Apc ( 1638T/1638T ) mice than in Apc (+/+) mice. Analyses using immunohistochemistry and electron microscopy indicated that follicular epithelial cells in Apc ( 1638T/1638T ) mice had an enlarged rough endoplasmic reticulum of irregular shape. These results suggest that the C-terminal domain of Apc is involved in thyroid morphology and function.

The adenomatous polyposis coli (APC) gene is mutated in familial adenomatous polyposis and in most sporadic colorectal tumors. During both embryonic and postnatal periods, APC is widely expressed in a variety of tissues, including the brain and gastrointestinal tract. The APC gene product (APC) is a large multidomain protein consisting of 2843 amino acids. APC downregulates the Wnt signaling pathway through its binding to beta-catenin and Axin. Most mutated APC proteins in colorectal tumors lack the beta-catenin-binding regions and fail to inhibit Wnt signaling, leading to the overproliferation of tumor cells. Several mouse models (APC580D, APCDelta716, APC1309, APCMin, APC1638T) have been established to investigate carcinogenesis caused by APC mutations. APC also binds to APC-stimulated guanine nucleotide exchange factor, the kinesin superfamily-associated protein 3, IQGAP1, microtubules, EB1, and discs large (DLG). APC has both nuclear localization signals and nuclear export signals in its molecule, suggesting its occasional nuclear localization and export of beta-catenin from the nucleus. APC is highly expressed in the intestinal and colorectal epithelia and may be involved in homeostasis of the enterocyte renewal phenomena, in which proliferation, migration, differentiation, and apoptosis are highly regulated both temporally and spatially. Through the many binding proteins mentioned, APC can exert multiple functions involved in epithelial homeostasis.