Expression of apoptosis-associated speck-like protein containing a caspase recruitment domain, a pyrin N-terminal homology domain-containing protein, in normal human tissues.

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is a pyrin N-terminal homology domain (PYD)- and caspase recruitment domain (CARD)-containing a proapoptotic molecule. This molecule has also been identified as a target of methylation-induced silencing (TMS)-1. We cloned the ASC cDNA by immunoscreening using an anti-ASC monoclonal antibody. In this study, we determined the binding site of the anti-ASC monoclonal antibody on ASC and analyzed the expression of ASC in normal human tissues. ASC expression was observed in anterior horn cells of the spinal cord, trophoblasts of the placental villi, tubule epithelium of the kidney, seminiferous tubules and Leydig cells of the testis, hepatocytes and interlobular bile ducts of the liver, squamous epithelial cells of the tonsil and skin, hair follicle, sebaceous and eccrine glands of the skin, and peripheral blood leukocytes. In the colon, ASC was detected in mature epithelial cells facing the luminal side rather than immature cells located deeper in the crypts. These observations indicate that high levels of ASC are abundantly expressed in epithelial cells and leukocytes, which are involved in host defense against external pathogens and in well-differentiated cells, the proliferation of which is regulated.

A new approach towards volumetric assessment of left ventricular function with MSCT.

Cardiovascular CT is considered the diagnostic standard for establishing the presence of a functional and dynamic imaging system. It is difficult, however, to estimate the ventricular motion and volumes that are processed using hundreds and thousands of CT images, in a few moments.The main concept and design of our work are two fold - the development of effective semi-automatic tools for measuring the sequential left ventricular volumes from the hundreds or thousands of cardiac trans-axial images, and providing a simple interface with an interactive diagnostic tool for the volumetry of left ventricle and valuable cardiac 4D visualisation.We converted ten and more sequential volume data sets of the heart acquired from retrospective ECG-gating helical scan into 3D images by volume rendering. These sequential 3D images could be displayed as a movie (4D cardiac image) file. Furthermore, we developed a method for semi-automatic calculation of ejection fraction (EF) and cardiac cycle (%)-volume (ml) curve for estimation of the motion and the volume of the left ventricle. This method involved the use an interactive selection tool in the region of interest (ROI). All 3D processing methods, such as, cutting objects, segmentation, and image fusion were based on mask processing data. We now describe the software developed for cardiac 4D imaging and the estimation of ventricular volume.

Pyrin N-terminal homology domain- and caspase recruitment domain-dependent oligomerization of ASC.

ASC was first identified as a caspase recruitment domain (CARD)-containing proapoptotic molecule that forms insoluble aggregates during apoptosis. Here, we report both the pyrin N-terminal homology domain (PYD) and CARD domains are involved in the aggregation of ASC. Preliminary experiments indicated that overexpression of ASC formed filament-like aggregates in COS-7 cells. Expression experiments using green fluorescent protein (GFP) constructs showed that not only the GFP-ASC-CARD but also the GFP-ASC-PYD formed filament-like aggregates in COS-7 cells. We confirmed these filament-like aggregates of both the ASC-PYD and the ASC-CARD due to homophilic interaction by immunoprecipitation method. We also demonstrated that the ASC-PYD associated with the ASC-CARD by heterophilic interaction. These observations suggest that the dimerization of the PYD as well as the CARD plays an important role in the oligomerization of ASC as an adaptor molecule.

Differential expression of moesin in cells of hematopoietic lineage and lymphatic systems.

Moesin is a member of the ERM family consisting of ezrin, radixin, and moesin. The protein is located in the plasma membrane similarly to ezrin and radixin, and is thought to regulate cellular movements and morphological changes. Using monoclonal antibody CR-22, the specificity of which against human moesin was confirmed by immunoprecipitation and western blotting analysis, we immunohistochemically stained various formalin-fixed and paraffin-embedded human tissues, in particular, clots of bone marrow and lymphatic tissues, to examine moesin expression in cells of hematopoietic lineage and lymphatic systems. In the bone marrow, moesin was expressed in myeloid cells, while little staining was detected in erythroid cells. Moesin was highly expressed in both the center and the periphery of mature megakaryocytes. In the lymphatic tissues, moesin was strongly expressed by T-lymphocytes in the paracortex. In the mantle zone, the periphery of the germinal center, moesin was expressed by small lymphocytes which were identified as B-lymphocytes. Furthermore, in areas of inflammation, moesin was expressed in both the center and the periphery of neutrophils, whereas in some neutrophils in distant areas, moesin was localized at the cellular periphery. These results suggest that differential expression of moesin in these cells is involved in their morphology and specialized functions.

Moesin and CD44 expression in cutaneous melanocytic tumours.

The ERM (ezrin, radixin and moesin) family members, located just beneath the plasma membranes, are thought to be involved in the association of action filaments with the plasma membrane. One of the family members, moesin, is reported to bind to CD44. Splice variants of CD44 are thought to be associated with tumour progression or differentiation. Our aim was to investigate immunohistochemically the expression of moesin together with CD44 on paraffin tissue sections of a series of melanocytic tumours. The material included 12 ordinary melanocytic naevi, six Spitz naevi, eight dysplastic naevi, six blue naevi, seven malignant melanomas in situ, 15 primary malignant melanomas, five metastatic melanomas to the skin and five lymph node metastases. In the normal skin and the melanocytic tumours the expression of moesin was largely similar to that of CD44 standard. Strong moesin staining was observed in benign melanocytic lesions and melanomas in situ. However, the expression was decreased in advanced malignant melanomas. The moesin labelling in melanoma cells was downregulated with the depth of dermal invasion. The immunoreactivity was also diminished in the skin metastases and the lymph node metastases of melanoma. These results suggest that in melanocytic tumours, the alternation in the expression of moesin may be involved in the progression of malignancy.

Expression of moesin and its associated molecule CD44 in epithelial skin tumors.

Moesin, one of the ERM (ezrin; radixin; moesin) family members, is directly associated with the cytoplasmic domain of CD44, which is now thought to be related to the metastatic potential of tumor cells. Using immunohistochemistry we investigated the expression of moesin in normal epidermis and various kinds of epithelial skin tumors: squamous cell carcinoma, verrucous carcinoma, Bowen's disease, solar keratosis, keratoacanthoma, basal cell carcinoma, and extramammary Paget's disease. Normal skin showed positive epidermal staining for moesin with the exception of the stratum corneum. The expression of moesin varied with the type of skin tumor. In basal cell carcinoma, Bowen's disease, and extramammary Paget's disease, moesin expression was either faint or negative. In contrast to Bowen's disease, invasive squamous cell carcinoma showed more intense and heterogeneous staining of the cytoplasm and the cell membrane. Verrucous carcinoma was weakly positive, with a tendency for the moesin to be distributed in the cell membrane. The staining pattern of moesin varied among the different kinds of epithelial skin tumors, and its expression was generally similar to that of the standard form of CD44. These results suggest that moesin is closely inter-related with CD44 in epithelial skin cells as seen in other cellular systems, and that the variable pattern of moesin staining among the skin tumor cells could reflect expression disorders associated with the transformation.

Murine ortholog of ASC, a CARD-containing protein, self-associates and exhibits restricted distribution in developing mouse embryos.

ASC (apoptosis-associated speck-like protein containing a CARD) was first identified as a cytosolic soluble protein that forms insoluble aggregates and enhances etoposide-induced apoptosis. We have cloned a murine ortholog of ASC (mASC) comprising 193 amino acids with a well-conserved pyrin N-terminal homology domain and caspase recruitment domain (CARD). mASC fused with green fluorescent protein appeared as a speck in transfected COS-7 cells and showed self-association. We analyzed mASC gene expression in developing embryos by in situ hybridization and found it to have a restricted distribution in mouse embryos. At E9.5, mASC was strongly expressed in the telencephalon, thalamic areas of the diencephalon, heart, and liver. Northern blotting analysis revealed that the mASC gene was expressed ubiquitously in multiple organs in adult mice. These findings indicate that mASC shows conservation of not only the primary structure of human ASC but also the ability to aggregate and has some similarity in its distribution to other CARD-containing molecules, including the apoptosis regulator Apaf-1.

Cytochrome c release into cytosol with subsequent caspase activation during warm ischemia in rat liver.

Apoptosis plays an important role in liver ischemia and reperfusion (I/R) injury. However, the molecular basis of apoptosis in I/R injury is poorly understood. The aims of this study were to ascertain when and how apoptotic signal transduction occurs in I/R injury. The apoptotic pathway in rats undergoing 90 min of warm ischemia with reperfusion was compared with that of rats undergoing prolonged ischemia alone. During ischemia, mitochondrial cytochrome c was released into the cytosol in a time-dependent manner in hepatocytes and sinusoidal endothelial cells, and caspase-3 and an inhibitor of caspase-activated DNase were cleaved. However, apoptotic manifestation and DNA fragmentation were not observed. After reperfusion, nuclear condensation, cells positive for terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling, and DNA fragmentation were observed and caspase-8 and Bid cleavage occurred. In contrast, prolonged ischemia alone induced necrosis rather than apoptosis. In summary, our results show that release of mitochondrial cytochrome c and caspase activation proceed during ischemia, although apoptosis is manifested after reperfusion.

La autoantigen is cleaved in the COOH terminus and loses the nuclear localization signal during apoptosis.

La autoantigen is a 47-kDa nuclear protein that binds to nascent polymerase III transcripts and a number of viral RNAs. We show that La protein was cleaved to generate a 43-kDa fragment during apoptosis of human leukemic HL-60 cells treated with camptothecin or etoposide. Immunofluorescence microscopy showed that the La protein level was increased in the cytoplasm during apoptosis of HL-60 cells. In addition, UV irradiation of HeLa cells led to the cleavage and redistribution of La protein upon apoptosis. Several lines of evidence show that La protein is cleaved by caspase-3 or closely related proteases at Asp-374 in the COOH terminus. When the full-length (La) and COOH-terminally truncated (La delta C374) forms of La protein were expressed as fusion proteins with green fluorescence protein (GFP), GFP-La delta C374 was predominantly cytoplasmic, whereas GFP-La was localized in the nucleus. These results suggest that La protein loses the nuclear localization signal residing in the COOH terminus upon cleavage and is thus redistributed to the cytoplasm during apoptosis.

ASC, a novel 22-kDa protein, aggregates during apoptosis of human promyelocytic leukemia HL-60 cells.

The cytoskeletal and/or nuclear matrix molecules responsible for morphological changes associated with apoptosis were identified using monoclonal antibodies (mAbs). We developed mAbs against Triton X-100-insoluble components of HL-60 cells pretreated with all-trans retinoic acid. In particular, one mAb recognized a 22-kDa protein that exhibited intriguing behavior by forming an aggregate and appearing as a speck during apoptosis induced by retinoic acid and other anti-tumor drugs. Cloning and sequencing of its cDNA revealed that this protein comprises 195 amino acids and that its C-terminal half has a caspase recruitment domain (CARD) motif, characteristic of numerous proteins involved in apoptotic signaling. We referred to this protein as ASC (apoptosis-associated speck-like protein containing a CARD). The ASC gene was mapped on chromosome 16p11.2-12. The antisense oligonucleotides of ASC were found to reduce the expression of ASC, and consequently, etoposide-mediated apoptosis of HL-60 cells was suppressed. Our results indicate that ASC is a novel member of the CARD-containing adaptor protein family.