野村 隆士

本学では、臨床検査技師教育課程において人体解剖実習を導入し、その有効かつ有益な教育方法を考察した。その結果、「心臓の構造を理解できた」と回答した学生は96%であり、他臓器においても80%以上と高率であった。また、医療職として必須の倫理的教育効果についても「ご遺体の尊厳についての理解」に関して96%の学生から肯定的な回答が得られた。一方で、教員の不足等による不満もあったが、今後は大学院生や勉学の意識の強い卒業生の実習参加などを通して、より広くかつ意義深い実習にしていきたいと考えている。(著者抄録)

Mesenchymal stem cells (MSCs) represent a promising cell source for stem cell therapy to replace neurons damaged by neurodegenerative diseases. A system designed for in vitro neuronal differentiation of MSCs is an indispensable technique, which provides MSC-derived functional neurons for cell-replacement therapies and valuable information in pre-clinical research. This study investigated the effects of reducing the volume of neural induction medium on cell viability and neural differentiation of MSCs. When MSCs were differentiated in low volumes of neural induction medium, rather than using the conventional method, the cell density on culture dishes significantly increased. The % cell death, including apoptosis and necrosis, was significantly lower in the lower volume method than in the conventional method. There were no significant differences between the lower volume and conventional methods in the expression levels of the neuronal marker genes. In an analysis of immunostaining for a mature neuronal marker, no significant difference was detected between the media volumes. These findings demonstrate that neuronal induction of MSCs in low volumes of differentiation medium promoted survival during differentiation and resulted in larger numbers of MSC-derived neurons, compared to the conventional method. This novel lower volume method offers both financial and cell-yield advantages over the conventional method.

The Dlg1 gene encodes a member of the MAGUK protein family involved in the polarization of epithelial cells. Null mutant mice for the Dlg1 gene (Dlg1(-/-) mice) exhibit respiratory failure and cyanosis, and die soon after birth. However, the cause of this neonatal lethality has not been determined. In the present study, we further examined Dlg1(-/-) mice and found severe defects in the cardiovascular system, including ventricular septal defect, persistent truncus arteriosus, and double outlet right ventricle, which would cause the neonatal lethality. These cardiovascular phenotypes resemble those of mutant mice lacking planar cell polarity (PCP) genes and support a recent notion that DLG1 is involved in the PCP pathway. We assessed the degree of involvement of DLG1 in the development of other organs, as the cochlea, intestine, and skeleton, in which PCP signaling has been suggested to play a role. In the organ of Corti, tissue elongation was inhibited accompanied by disorganized arrangement of the hair cell rows, while the orientation of the stereocilia bundle was normal. In the sternum, cleft sternum, abnormal calcification pattern of cartilage, and disorganization of chondrocytes were observed. Furthermore, shortening of the intestine, sternum, and long bones of the limbs was observed. These phenotypes of Dlg1(-/-) mice involving cellular disorganization and insufficient tissue elongation strongly suggest a defect in the convergent extension movements in these mice. Thus, our present results provide a possibility that DLG1 is particularly required for convergent extension among PCP signaling-dependent processes.

In order to elucidate the function of anti-aromatic acid decarboxylase (AADC)-only-positive cells in the alimentary canal, 5-hydroxy-L-tryptophan (5-HTP) or L-3,4-dihydroxyphenylalanine (L-DOPA) was intraperitoneally injected into the laboratory shrew, Suncus murinus, and immunohistochemical studies were conducted on continuous sections of the alimentary canal using specific antisera against tyrosine hydroxylase (TH), AADC, dopamine (DA), and serotonin (5-HT). AADC-only-positive cells localized to the epithelial layer of the alimentary canal from the stomach to the large intestine. These AADC-only-positive cells became DA- and AADC-positive cells after L-DOPA injection, and 5-HT- and AADC-positive cells after 5-HTP injection. These results strongly indicate that the AADC-only-positive cells in the alimentary canal of Suncus murinus are capable of synthesizing DA and 5-HT simultaneously upon administration of L-DOPA and 5-HTP.

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 Apc(1638T/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 Apc(1638T/1638T) mice. Apc(1638T/1638T) mice showed abnormal morphology of the dendritic spines and impaired long-term potentiation of synaptic transmission in the hippocampal CA1 region. Moreover, Apc(1638T/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.

Lymphocyte enhancer factor 1 (LEF1), a member of the LEF/T-cell-specific factor (TCF) family of the high mobility group domain transcription factors, acts downstream in canonical Wnt signaling. Aberrant transactivation of LEF1 contributes to the tumorigenesis of colonic neoplasms, sebaceous skin tumors, and lymphoblastic leukemia. LEF1-associated proteins are crucial for regulating its transcriptional activity. In this study, glutathione-S-transferase pull-down assay and mass spectrometry enabled identification of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) as a novel interaction partner for LEF1. The interaction between LEF1 and DNA-PKcs was confirmed using in vivo co-immunoprecipitation. Furthermore, double immunofluorescence observations showed that LEF1 and DNA-PKcs colocalized in the nuclei of colon adenocarcinoma cell lines. Identification of the interaction between LEF1 and DNA-PKcs may provide clues for a novel therapy for cancer treatment as well as for understanding LEF1-mediated transcriptional regulation.

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.

Calreticulin (CRT)-1 is a major Ca2+-buffering protein in the lumen of the endoplasmic reticulum. Human and murine CRT-2 was isolated in 2002, but the subcellular localization and function is still unclear. Here, we studied the intracellular localization and function of CRT-2 with hemagglutinin-tagged (HA-) human CRT-2. Western blotting revealed HA-CRT-2 as a single band at 50 kDa. Using immunofluorescence microscopy of cultured fibroblasts and epithelial cells transfected with HA-CRT-2 cDNA, labeling for HA-CRT-2 was seen as a reticular network with a nuclear envelope pattern that colocalized with calnexin and protein disulfide isomerase. Immunoelectron microscopy confirmed that HA-CRT-2 was localized in the lumen of the endoplasmic reticulum. Stains-all staining, a method to detect Ca2+-binding proteins, could not stain the immunoprecipitate of HA-CRT-2, although HA-CRT-1 immunoprecipitate was stained blue. These results indicate that the molecular weight of the non-tagged CRT-2 on SDS-PAGE is 49 kDa, and that CRT-2, as well as CRT-1, is localized in the lumen of the endoplasmic reticulum, but that CRT-2 capacity for Ca2+-binding may be absent or much lower than that of CRT-1.

ICAT, inhibitor of (beta-catenin and T cell factor, or CtnnbipI, is a negative regulator of the Wnt signaling pathway that interferes with the interaction between beta-catenin and T cell factor. Some ICAT-deficient (ICAT(-/-)) embryos exhibit unilateral or bilateral renal agenesis. In this study, we investigated developmental processes in the ICAT-/- kidney. ICAT was highly expressed in both the ureteric bud (UB) and the surrounding metanephric mesenchymal (MM) cells in the metanephros of embryonic day E11.5-E13.5 wild-type (ICAT(+/+)) mouse. In the E12.5-ICAT(-/-) metanephros, UB branching was delayed, and a T-shaped, bifurcated UB was frequently seen; this was never seen in the E12.5-ICAT(+/+) metanephros. More apoptotic MM cells were detected in the ICAT-/- metanephros than in the ICAT+/+ metanephros. These results suggest that the loss of ICAT gene function causes the arrest of UB branching and the apoptotic death of MM cells, resulting in renal agenesis. (C) 2007 Elsevier Inc. All rights reserved.

Mutations in the adenomatous polyposis coli (APC) gene are associated with familial adenomatous polyposis and sporadic colorectal tumours. The APC gene is expressed ubiquitously in various tissues, especially throughout the large intestine and central nervous system (CNS). In the CNS, the expression of the APC protein is highest during embryonic and early postnatal development. APC associates through its C-terminal region with postsynaptic density (PSD)-95, a neuronal protein that participates in synapse development. Here, we examined the involvement of APC in synaptogenesis. In cultured hippocampal neurons, both overexpression of a dominant-negative construct that disrupts the APC-PSD-95 interaction and knockdown of APC expression using small interfering RNA (siRNA) inhibited the clustering of PSD-95 and a glutamate receptor subunit, and reduced alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA)-induced activity of AMPA receptors however, the clustering of an N-methyl-d-aspartate (NMDA) receptor subunit was unaffected. These results are suggestive of APC involvement in the development of glutamatergic synapses. © The Authors (2007).

A mouse monoclonal antibody (G9, Horio et al. in Cell Motil Cytoskel 44:284-295, 1999) that was raised against the gamma-tubulin from a fission yeast, Schizosaccharomyces pombe, showed a unique staining in the mouse small intestine. Similar to another anti-gamma-tubulin antibody that is commercially available, G9 showed typical dot-like staining corresponding to the microtubule-organizing center in the free cells of the epithelium and the connective tissue under it. In addition, G9 stained the cell-cell contacts in the epithelium. This stained region was not bicellular but tricellular junctions of the enterocytes. This staining was unique to G9 and was diminished on the sample of the mouse small intestine, which had lost most of its filamentous microtubules through the preparation process. The tricellular junction is thought to be the weakest point of the epithelial barrier, and no other junctional structures have been identified except for the central sealing elements extending from the tight junctions between the two cells. Our results suggest the existence of a new molecule underlying the tricellular junctions, which may relate to gamma-tubulin and the microtubules.

Caveolae have been reported to contain a number of proteins related to Ca2+ mobilization, including an inositol 1,4,5-trisphosphate receptor-like protein, and a plasmalemmal calcium pump. In search of other Ca2+-related proteins, we found that a polyclonal antibody to calreticulin (CRT), an endoplasmic reticulum (ER) Ca2+-binding protein, binds to the cytoplasmic surface of caveolae. By immunofluorescence microscopy of cultured fibroblasts, labeling by the anti-CRT antibody was found colocalized with caveolin-1. Immunogold electron microscopy of ultrathin frozen sections showed that the antibody decorates the cytoplasmic surface of caveolae. The caveolar labeling could be due to CRT that leaked out of the ER, but the labeling persisted even when isolated plasmalemmal preparations were treated with 10 mM EDTA, 1 M NaCl, or 0.1 M Na2CO3 (pH 11). Furthermore, in SDS-digested freeze-fracture replicas, labeling was associated with caveolae in the P face of the plasma membrane. However, in cells transfected with hemagglutinin (HA)-tagged cDNA, the labeling by anti-HA was observed only in the ER and was not found in caveolae. Taken together, the results suggest that a protein structurally similar to CRT is localized in caveolae as an integral membrane protein, and that the protein could be functionally related to the intracellular Ca2+ homeostasis.

CD13, a receptor for human coronavirus 229E (HCoV-229E), was identified as a major component of the Triton X-100-resistant membrane microdomain in human fibroblasts. The incubation of living fibroblasts with an anti-CD13 antibody on ice gave punctate labeling that was evenly distributed on the cell surface, but raising the temperature to 37degreesC before fixation caused aggregation of the labeling. The aggregated labeling of CD13 colocalized with caveolin-1 in most cells. The HCoV-229E virus particle showed a binding and redistribution pattern that was similar to that caused by the anti-CD13 antibody: the virus bound to the cell evenly when incubated on ice but became colocalized with caveolin-1 at 37degreesC; importantly, the virus also caused sequestration of CD13 to the caveolin-1-positive area. Electron microscopy confirmed that HCoV-229E was localized near or at the orifice of caveolae after incubation at 37degreesC. The depletion of plasmalemmal cholesterol with methyl beta-cyclodextrin significantly reduced the HCoV-229E redistribution and subsequent infection. A caveolin-1 knockdown by RNA interference also reduced the HCoV-229E infection considerably. The results indicate that HCoV-229E first binds to CD13 in the Triton X-100-resistant microdomain, then clusters CD13 by cross-linking, and thereby reaches the caveolar region before entering cells.

Mice aged 1, 4 or 8 weeks were inoculated with haemagglutinating encephalomyelitis virus (HEV), strain 67N, by the intracerebral (i.c.), intranasal (i.n.), intraperitoneal (i.p.), subcutaneous (s.c.), intravenous (i.v.) or oral route, with different doses. In I-week-old mice, mortality and mean time to death were mostly the same regardless of the inoculation route, except for the oral route, which appeared to be the least effective. The virus killed 4-week-old mice readily by all routes of inoculation except the oral, and 8-week-old mice by i.c., i.n. or s.c. inoculation. In descending order of efficacy, the routes of HEV infection were: i.c., i.n., s.c., i.p., i.v. and oral. To follow the spread of HEV from peripheral nerves to the central nervous system (CNS), the virus was inoculated subcutaneously into the right hind leg of 4-week-old mice. The virus was first detected in the spinal cord on day 2, and in the brain on day 3. The brain titres became higher than those of the spinal cord, reaching a maximum of 10(7)PFU/0.2g when the animals were showing CNS signs. Viral antigen was first detected immunohistochemically in the lumbar spinal cord and the dorsal root ganglion ipsilateral to the inoculated leg; it was detected later in the pyramidal cells of the hippocampus and cerebral cortex, and in the Purkinje cells of the cerebellum but not in the ependymal cells, choroid plexus cells or other glial cells. The infected neurons showed no cytopathological changes. (C) 2003 Elsevier Ltd. All rights reserved.

In the current study, we examined the cytoskeletal architecture of cod hepatic stellate cells. We found that the cod hepatic stellate cells contain abundant cytoplasmic filaments. Deep-etch electron microscopy showed that the major component of the cytoplasmic filaments was intermediate filaments, although microtubules and microfilaments were also found in the cytoplasmic filament bundles. Immunoelectron microscopy revealed the presence of βtubulin, α smooth muscle actin, smooth muscle type myosin, desmin and cytokeratin but not vimentin or glial fibrillar acidic protein. These results demonstrate that the cytoplasmic filaments of cod hepatic stellate cells are composed of desmin and cytokeratin intermediate filaments, acto-myosin complexes and microtubules, suggesting that the cod hepatic stellate cells have both contractile and structural functions. The expression of cytokeratin in cod hepatic stellate cells indicates that they serve for mechanical support in the extremely soft liver tissues of cods with their abundant lipids.

As undifferentiated precursor cells in the CNS, neural progenitor cells (NPCs) supply new neurons and glial cells to repair damage within the adult brain. Recently, NPCs were found to undergo apoptosis. In serum-free basic fibroblast growth factor-containing culture medium, primary culture cells from fetal mouse neuroepithelium expressed nestin, and thus might be regarded as NPCs. These NPCs demonstrated apoptosis under electron microscopy and TUNEL assay. Treatment of NPCs with lithium, a specific inhibitor of glycogen synthase kinase 3beta (GSK3beta), significantly suppressed apoptosis. Activity of pro-apoptotic protease caspase-3 was not detected in either lithium-untreated or-treated NPCs. These findings suggest that lithium may protect NPCs against apoptosis by inhibiting caspase-3-independent apoptotic pathways.

The relationship between caveolin-1 isoforms (alpha and beta) and caveolar ultrastructure was studied, By immunofluorescence microscopy of human fibroblasts, caveolae were observed as dots positive for caveolin-1, but many dots labeled by an antibody recognizing both isoforms (anti-alpha beta) were not labeled hy another antibody specific for the alpha isoform (anti-alpha), Immunogold electron microscopy of freeze-fracture replicas revealed caveolae of different depths, and indicated that anti-ct labeled deep caveolae preferentially over shallow ones, whereas anti-ap labeled both forms with an equivalent frequency and intensity, The presence of the beta isoform in deep caveolae was confirmed by labeling elpitope-tagged beta -caveolin When made to be expressed in HepG2 cells lacking endogenous caveolins, the alpha isoform formed caveolar depressions efficiently, but the beta isoform hardly did so. Caveolae were also formed in cells expressing the two isoforms, but their frequency was variable among cells of the same clone. Coexpression of caveolin-1 and caveolin-2 caused more efficient formation of deep caveolae than caveolin-1 alone, The result indicates that the two isoforms of caveolin-1 have a different potential for forming caveolae structure, and more importantly, that deep and shallow caveolae may be diversified in their molecular composition.

Caveolin-1, a scaffolding protein of caveolae, is known to be tyrosine-phosphorylated by Src kinases. Recently we generated a specific antibody to caveolin-1 phosphorylated at tyrosine-14 (PY14) (R. Nomura and T. Fujimoto, 1999, Mol. Biol. Cell 10, 975-986). In the present study, by applying PY14 to sections of normal rat tissues, we found that tyrosine phosphorylation of caveolin-1 occurred in limited locations, including the endothelium of the continuous capillaries and small venules. Cultured endothelial cells were not labeled by PY14 under a standard culture condition, but became positively labeled when exposed to oxidative stresses and/or tyrosine phosphatase inhibitors. The reaction was prohibited by pretreating the cells with herbimycin A or genistein. Vasoactive reagents or physical stimuli did not cause the phosphorylation. Concomitant with the tyrosine phosphorylation, the number of invaginated caveolae decreased drastically, and vesicles labeled intensely for caveolin-1 appeared in the cytoplasm; the average diameter of the vesicles was larger than that of caveolae. The result implies that tyrosine phosphorylation of caveolin-1 occurs at tyrosine-14 in the normal rat endothelium in vivo and may induce caveolar vesiculation and/or fusion. (C) 1999 Academic Press.

Caveolin-1 was discovered as a major substrate for v-Src, but the effect of its tyrosine phosphorylation has not been known. We generated a specific antibody (PY14) to caveolin-1 phosphorylated at tyrosine 14 and studied the significance of the modification. By Western blotting of lysates of v-Src-expressing cells, PY14 recognized not only a 22-kDa band (the position of nonphosphorylated caveolin-1) but bands at 23-24 and 25 kDa. Bands of slower mobility were diminished by dephosphorylation and were also observed for mutant caveolin-1 lacking tyrosine 14. By immunofluorescence microscopy, PY14 did not label normal cells but detected large dots in v-Src-expressing cells. Immunoelectron microscopy revealed that the dots corresponded to aggregated caveolae and/or vesicles of various sizes; besides, the label was observed in intramembrane particle-free areas in the plasma membrane, which appeared to have been formed by fusion of flattened caveolae. A positive reaction with PY14 was found in normal cells after vanadate or pervanadate treatment; it occurred mainly at 22 kDa by Western blotting and was not seen as large dots by immunofluorescence microscopy. Detergent solubility, oligomerization, and association with caveolin-2 were observed similarly for caveolin-1 in normal and v-Src-expressing cells. The results indicate that phosphorylation of caveolin-1 in v-Src-expressing cells occurs at multiple residues and induces flattening, aggregation, and fusion of caveolae and/or caveolae-derived vesicles.

Caveolae in the plasma membrane have been a focus of intensive research during the past several years. There has been confusion concerning caveolae and caveola-like membrane domains, but it is now generally thought that the latter is a region distinct from caveolae. However, due to similar buoyancy of caveolae and caveola-like membranes, whether caveolae in situ are enriched with a given molecule is often difficult to be concluded by biochemical techniques alone. Furthermore, relatively shallow caveolae may be detected by some techniques, but not by others. Thus whether a molecule is enriched in caveolae should be confirmed by methods based on different principles. Among many putative caveolar molecules, those related to Ca2+ influx and extrusion were shown to be concentrated in caveolae by both immunocytochemical and biochemical techniques. In conjunction with other characteristics, the result implies that caveolae may function as a mobile compartment for Ca2+ signalling.

Various factors influencing plaque formation of Chuzan virus in BHK-21 cell monolayers were studied and a practical method for plaque assay was developed. On addition of trypsin (5 mu g/ml) and/or diethylaminoethyl (DEAE)-dextran (50 mu g/ml) to the virus diluent as the virus adsorption medium and agar overlay medium, the number of plaques increased. When 100 mu g/ml DEAE-dextran was added to the diluent and overlay medium, plaques were produced in about 10-fold higher numbers than without trypsin and DEAE-dextran. Based on these results, a practical plaque assay method for Chuzan virus was established. Using this method, one-step growth of Chuzan virus was performed at an input multiplicity of 25 plaque-forming units (PFU) per cell. Cytopathic effects were first observed at 7.5 h post-inoculation (p.i.), and were complete at 12 h p.i. The titre of cell-associated virus, after gradual decline during the first 3 h of incubation, showed a rise within 4.5 h p.i. and a rise to a plateau of 10(6.3)PFU/0.2 ml at 12 h p.i. By indirect immunofluorescence, virus-specific antigen was detected in the cytoplasm of the cells at 4.5 h p.i., and all the cells fluoresced at 6 h p.i. Haemagglutination activity was first detected in infected whole cultures at 7.5 h p.i. reaching a plateau of 1:64 at 15 h p.i. Plaque formation and haemagglutination by the virus were specifically inhibited by antisera against the original and the plaque-cloned virus.

The outbreak of sialoadenitis occurred in a laboratory rat colony and the causative agent was isolated from the affected salivary glands of diseased rats using the established cell line LBC. The isolate readily multiplied, producing clear cytopathic effects with syncytium formation, and it was identified virologically and serologically as rat sialodacryoadenitis virus. In attempts to isolate the virus by primary rat kidney (PRK) cells and suckling mice as well as LBC cells, the LBC cells showed higher susceptibility for the virus growth as compared with TRK cells or the brain of suckling mice. The isolation rate of virus was 100% (5/5) in LBC, 40% (2/5) in PRK cells and 60% (3/5) in suckling mice. After four passages in the LBC cells, the virus did not produce disease in adult rats, while the mouse brain-passaged virus did.

Bubble-free thin sections of porous specimens, such as pumices and accretionary lapilli, were made by mean of vacum treatment.

The preparation- of defatted mustard by extraction with supercritical carbon dioxide (SC-C02is described. Oil was not extracted from native mustard seeds but was extracted from compressed mustard seeds with SC-C02 at 300 atm and 40°C. When the extraction time and temperature were kept constant at 3 hr and 40°C, 80~90% of the oil in the compressed mustard seeds could be extracted with SC-C02 at more than 300 atm. The solubility of the mustard seed oil in SC-C02 at 300 atm and 40°C was estimated to be about 0.48%. The synigrin content and myrosinase activity in the defatted mustard seeds with SC-C02 at 300 atm and 40°C for 3 hr were comparable to those in native seeds. Thus, the extraction with SC-C02 made it possible to prepare defatted mustard of high quality without decreasing the synigrin content and myrosinase activity. © 1987, Japan Society for Bioscience, Biotechnology, and Agrochemistry. All rights reserved.