In situ visualization of plasma cells producing antibodies reactive to Porphyromonas gingivalis in periodontitis: the application of the enzyme-labeled antigen method.

Porphyromonas gingivalis is a keystone periodontal pathogen. Histologocally, the gingival tissue in periodontitis shows dense infiltration of plasma cells. However, antigens recognized by antibodies secreted from the immunocytes remain unknown. The enzyme-labeled antigen method was applied to detecting plasma cells producing P. gingivalis-specific antibodies in biopsied gingival tissue of periodontitis. N-terminally biotinylated P. gingivalis antigens, Ag53 and four gingipain domains (Arg-pro, Arg-hgp, Lys-pro and Lys-hgp) were prepared by the cell-free protein synthesis system using wheatgerm extract. With these five labeled proteins as probes, 20 lesions of periodontitis were evaluated. With the AlphaScreen method, antibodies against any one of the five P. gingivalis antigens were detected in 11 (55%) serum samples and 17 (85%) tissue extracts. Using the enzyme-labeled antigen method on paraformaldehyde-fixed frozen sections of gingival tissue, plasma cells were labeled with any one of the five antigens in 17 (94%) of 18 specimens, in which evaluable plasma cells were detected. The positivity rates in periodontitis were significantly higher than those found previously in radicular cysts (20% in sera and 33% in tissue extracts with the AlphaScreen method, and 25% with the enzyme-labeled antigen method). Our findings directly indicate that antibodies reactive to P. gingivalis are locally produced in the gingival lesions, and that inflammatory reactions against P. gingivalis are involved in periodontitis.

In vivo eradication of MLL/ENL leukemia cells by NK cells in the absence of adaptive immunity.

It remains unclear how the immune system affects leukemia development. To clarify the significance of the presence of immune systems in leukemia development, we transferred MLL/ENL leukemia cells into immune-competent or immune-deficient mice without any preconditioning including irradiation. The wild-type mice did not develop leukemia, whereas all the Rag2(-/-)γc(-/-) mice lacking both adaptive immune cells and natural killer (NK) cells developed leukemia, indicating that leukemia cells were immunologically rejected. Interestingly, leukemia cells were also rejected in 60% of the Rag2(-/-) mice that lacked adaptive immune cells but possessed NK cells, suggesting that NK cells play a substantial role in the rejection of leukemia. Moreover, engraftment of leukemia cells was enhanced by NK cell depletion in Rag2(-/-) recipients and inhibited by transfer of NK cells into Rag2(-/-)γc(-/-) recipients. Upregulation of NKG2D (NK group 2, member D) ligands in MLL/ENL leukemia cells caused elimination of leukemia cells by NK cells. Finally, we found that leukemia cells resistant to elimination by NK cells had been selected during leukemia development in Rag2(-/-) recipients. These results demonstrate that NK cells can eradicate MLL/ENL leukemia cells in vivo in the absence of adaptive immunity, thus suggesting that NK cells can play a potent role in immunosurveillance against leukemia.

CD138-negative clonogenic cells are plasma cells but not B cells in some multiple myeloma patients.

Clonogenic multiple myeloma (MM) cells reportedly lacked expression of plasma cell marker CD138. It was also shown that CD19(+) clonotypic B cells can serve as MM progenitor cells in some patients. However, it is unclear whether CD138-negative clonogenic MM plasma cells are identical to clonotypic CD19(+) B cells. We found that in vitro MM colony-forming cells were enriched in CD138(-)CD19(-)CD38(++) plasma cells, while CD19(+) B cells never formed MM colonies in 16 samples examined in this study. We next used the SCID-rab model, which enables engraftment of human MM in vivo. CD138(-)CD19(-)CD38(++) plasma cells engrafted in this model rapidly propagated MM in 3 out of 9 cases, while no engraftment of CD19(+) B cells was detected. In 4 out of 9 cases, CD138(+) plasma cells propagated MM, although more slowly than CD138(-) cells. Finally, we transplanted CD19(+) B cells from 13 MM patients into NOD/SCID IL2Rγc(-/-) mice, but MM did not develop. These results suggest that at least in some MM patients CD138-negative clonogenic cells are plasma cells rather than B cells, and that MM plasma cells including CD138(-) and CD138(+) cells have the potential to propagate MM clones in vivo in the absence of CD19(+) B cells.

Histopathological diagnosis of Japanese spotted fever using formalin-fixed, paraffin-embedded skin biopsy specimens: usefulness of immunohistochemistry and real-time PCR analysis.

Japanese spotted fever (JSF) is caused by Rickettsia japonica, and lethal cases are reported yearly in southwest Japan. We thus established the method of diagnosing JSF by immunohistochemistry (IHC) and real-time PCR (RT-PCR) using formalin-fixed, paraffin-embedded skin biopsy specimens. Two monoclonal antibodies were used for IHC, and the 17k genus common antigen gene served as the target of RT-PCR. We collected skin biopsy (n = 61) and autopsy (n = 1) specimens from 50 patients clinically suspected of JSF. Immunohistochemically, the rickettsial antigens were localized as coarse dots in the cytoplasm of endothelial cells and macrophages. Thirty-one seropositive cases plus one autopsy case (group A) and nine seronegative cases but with positive IHC and/or RT-PCR (group B) were judged as JSF. Nine cases were regarded as non-JSF disorders based on negative serology, IHC and RT-PCR (group C). Of 50 biopsies (eschar 34, eruptions 10, and scabs 6) from groups A and B, IHC and RT-PCR positivities were 94% (32/34) and 62% (21/34) for eschar, 80% (8/10) and 30% (3/10) for eruptions, and 33% (2/6) and 50% (3/6) for scabs. For IHC, eschar was most suitable, and scabs were insufficient. Unexpectedly, 18 biopsies happened to be fixed in 100% formalin, and this lowered the detection rate by RT-PCR, but IHC was tolerant. Sequence analysis using five skin biopsy specimens confirmed a 114 bp DNA stretch homologous to that reported for the target gene of R. japonica. In 26 (84%) of the 31 seropositive patients, the diagnosis was made by IHC and/or RT-PCR earlier than serology.

Low-temperature synthesis of single-walled carbon nanotubes by alcohol gas source growth in high vacuum.

Carbon nanotube (CNT) growth was carried out on SiO2/Si substrates with a Co catalyst using an alcohol gas source method in an ultra-high vacuum chamber. The resulting CNTs were characterized by scanning electron microscopy (SEM), Raman spectroscopy and transmission electron microscopy (TEM). Reducing the ethanol pressure decreased the optimum growth temperature for maximum yield, enabling single-walled carbon nanotube (SWNT) growth at 400 degrees C. By employing an Al2Ox buffer layer, SWNT yield increased several times, even at 400 degrees C. Under TEM observation, the Co particle size on the Al2Ox layers did not show a significant dependence on the growth temperature between 400 and 700 degrees C. Raman and TEM results confirmed activation of Co particles with larger diameter (>1 nm) by the Al2Ox buffer layer.

Antineutrophil cytoplasmic antibody-associated vasculitis with oculomotor nerve palsy.

We report a patient with antineutrophil cytoplasmic antibody-associated vasculitis with oculomotor nerve palsy. The patient presented with a high fever, diplopia, blepharoptosis and impairment of ocular movement of the left eye except for lateral gaze. Multiple erythematous and livedoid lesions were observed on the forehead, both cheeks and both legs. Laboratory examination showed positive results for myeloperoxidase antineutrophil cytoplasmic antibodies. Skin biopsy revealed leucocytoclastic vasculitis of the small arteries in the lower dermis. The patient was successfully treated with systemic corticosteroids.

Conjunctival displacement to the corneal side for oblique-parallel insertion in 25-gauge vitrectomy.

PURPOSE: To assess the usefulness of the method of oblique-parallel trocar insertion with conjunctival displacement to the corneal side in 25-gauge (G) transconjunctival vitrectomy. METHODS: 25-G vitrectomy was performed in 77 consecutive eyes. Before making obliqueparallel trocar insertions, the conjunctiva was conventionally displaced superiorly in 35 eyes, but was displaced toward the corneal side in 42 eyes. After surgery, the distance between the scleral and conjunctival wounds was measured with calipers. The frequency of scleral wound exposure was assessed. RESULTS: After cannula removal at the end of surgery, inferior repositioning of the superiorly displaced conjunctiva was observed, while marked posterior repositioning of the corneal side caused displacement of the conjunctiva due to gravity. The superior displacement distances between the sclera and conjunctival wounds were 2.4+/-0.3 mm at the infusion port, 2.0+/-0.4 mm at the superior temporal port, and 1.9+/-0.4 mm at the superior nasal port, while the corresponding distances for corneal side displacement were 3.6+/-0.5, 3.5+/-0.5, and 2.5+/-0.5mm, and were all significantly (p<0.0001) greater with corneal side displacement. The frequency of scleral wound exposure due to conjunctival damage around the cannula (infusion port) was significantly (p=0.0164) lower for corneal side displacement (0/42; 16.7%) than superior displacement (5/35; 14.3%). There was no postoperative endophthalmitis in all 77 patients studied. CONCLUSIONS: In 25-G transconjunctival vitrectomy, using oblique-parallel trocar insertions with the conjunctiva displaced toward the corneal side results in marked posterior repositioning of the conjunctiva after cannula extraction. Corneal side conjunctival displacement is technically easy and completely covers the scleral wound. This method is expected to be effective in preventing endophthalmitis.

25-Gauge peripheral iridectomy during vitrectomy.

PURPOSE: To report peripheral iridectomy using a 25-gauge vitreous cutter in a 42-year-old man with pupillary block due to adhesion of the internal iris surface to the continuous circular capsulorhexis. METHODS: A corneal opening was made at 10 o'clock during vitrectomy. A 25-gauge vitreous cutter was inserted into the anterior chamber with the port facing downward, and peripheral iridectomy at the 12 o'clock position was performed. The vitreous cutter was set at a cutting speed of 2500 cpm and the aspiration pressure at 600 mmHg. RESULTS: A 25-gauge vitreous cutter with a fine shaft could easily be inserted into the peripheral anterior chamber, and there was no contact with the corneal endothelium even when the anterior chamber became shallow in association with iridectomy. In this patient, pupillary block resolved with peripheral iridectomy, and ocular pressure was also controlled. CONCLUSIONS: 25-gauge peripheral iridectomy is a simple technique that permits iridectomy of appropriate size at any desirable location.

Can we develop biomarkers that predict response of cancer patients to immunotherapy?

PRIMARY OBJECTIVE: The primary objective is to delineate the potential utility of cancer biomarkers that correlate and predict response to immunotherapy in cancer patients who are refractory to conventional therapeutics. Unlike significant development of biomarkers that predict response to chemotherapy, very few biomarkers have been developed to predict the response to immunotherapy. MAIN OUTCOMES AND RESULTS: This article describes briefly the importance of characterizing and validating biomarkers for immunotherapy. A few examples have been provided, such as the transcription factor NF-kappaB, the transcription repressor Yin-Yang 1 (YY1), the pro-apoptotic gene product (Smac/DIABLO) and the circulating Fas and Fas ligand. These biomarkers have been determined to be of prognostic significance in different cancers. CONCLUSIONS: Immunotherapy is considered as an alternative therapy in the treatment of cancer patients who are refractory to chemotherapy/radiation/hormonal therapies. Cross-resistance to apoptosis develops between cancer cells that are resistant to conventional therapeutics and immunotherapy. Therefore, it is important to develop biomarkers that will determine patient response to immunotherapy.

Carbonate-containing hydroxyapatite derived from calcium tripolyphosphate gel with urea.

Carbonate containing hydroxyapatite (CO3HAp) is one of the candidate materials as a bioresorbable bone substitute. In the present work, CO3HAp was efficiently prepared by a hydrothermal treatment of calcium tripolyphosphate gel with urea at 140 degrees C for 24 h. Chemical potential plots of the CO3HAp for estimation of its dissolution behavior suggested that the CO3HAp is more soluble than hydroxyapatite (HAp) and is as soluble as octacalcium phosphate (OCP) and/or beta -tricalcium phosphate (TCP). This material is expected to be applied to bioresorbable materials such as bone fillers.

Immunohistochemical and ultrastructural findings related to the blood--brain barrier in the blood vessels of the cerebral white matter in aged dogs.

Immunohistochemical and ultrastructural analysis of canine brain tissue was performed to determine whether cerebral capillaries, which form the blood--brain barrier (BBB), display age-related morphological changes in the white matter (WM). A slight decrease in laminin immunolabelling was detected in the basement membranes (BMs) of capillaries in the WM of old dogs, as compared with that in the brains of young dogs. The Prussian blue DAB post-DAB enhancement method detected iron present in macrophages and astrocytes in the WM. Copper/zinc superoxide dismutase, MT-I and -II and MT-III immunoreactivity was detected mainly in reactive astrocytes in the WM of aged dogs. Ultrastructurally, collagen-like fibrils were detected to a variable degree in the spaces between the BMs of capillary endothelial cells and astrocytes in the WM of some aged dogs. These results suggest that age-related morphological changes in capillaries of the WM are associated with BBB dysfunction, leading to the exudation of serum constituents, including harmful substances (e.g., iron), thereby causing tissue damage by oxidative injury. These factors may play a role in the pathogenesis of severe degenerative changes in the WM of aged dogs.

Significance of dihydropyrimidine dehydrogenase activity in renal cell carcinoma.

Dihydropyrimidine dehydrogenase (DPD) is the rate-limiting enzyme in the pathway of uracil and thymine catabolism. DPD is also the principal enzyme involved in the degradation of 5-fluorouracil (5-FU), an anticancer chemotherapeutic agent that is used clinically to treat renal cell carcinoma (RCC). Little is known about the significance of DPD activity in human cancers. We investigated the activity of DPD in 68 RCC and the relationship between DPD activity and the sensitivity to 5-FU. The levels of DPD activity in RCC and normal kidney samples were determined by the 5-FU degradation assay. The sensitivity to 5-FU was assessed by the microculture tetrazolium dye assay. The activity of DPD was approximately 2-fold higher in normal kidney compared with RCC. DPD activity in Stage I/II RCC was approximately 2-fold higher than that in Stage III/IV RCC. The levels of DPD activity in Grade 1 and Grade 2 RCC were 3 and 2-fold higher, respectively, than that in the Grade 3 cancers. There was an inverse correlation between DPD activity in RCC cells and their sensitivity to 5-FU. Furthermore, 5-chloro-2,4-dihydroxypyridine (CDHP), a potent DPD inhibitor, enhanced the sensitivity to 5-FU. The current study is the first to demonstrate that the level of DPD activity was inversel correlated with both the progression of the disease and increased grade of RCC, and that DPD activity was inversely associated with the sensitivity of RCC to 5-FU, which was enhanced by a DPD inhibitor. These results suggest that a low DPD activity may be associated with the malignant potential of RCC. In addition, it may be possible to overcome 5-FU resistance by using DPD inhibitors in the treatment protocols of 5-FU-based chemotherapy for RCC.

Resveratrol and cancer: chemoprevention, apoptosis, and chemo-immunosensitizing activities.

The polyphenolic compound Resveratrol is a naturally occurring phytochemical and can be found in many plant species, including grapes, peanuts and various herbs. Several studies have established that Resveratrol can exert anti-oxidant and anti-inflammatory activities. It also has activity in the regulation of multiple cellular events associated with carcinogenesis. This review describes the general properties of Resveratrol including its relationship to estrogen, its effect on lipid metabolism, its cardiovascular effects, and its role on gene expression. Resveratrol has also been examined in several model systems for its potential effect against cancer. Its anti-cancer effects include its role as a chemopreventive agent, its ability to inhibit cell proliferation, its direct effect in cytotoxicity by induction of apoptosis and on its potential therapeutic effect in pre-clinical studies. In addition, Resveratrol has been shown to exert sensitization effects on cancer cells that will result in a synergistic cytotoxic activity when Resveratrol is used in combination with cytotoxic drugs in drug-resistant tumor cells. Clearly, the studies with Resveratrol provide support for the use of Resveratrol in human cancer chemoprevention and combination with chemotherapeutic drugs or cytotoxic factors in the treatment of drug refractory tumor cells.

Nonviral genetic transfer of Fas ligand induced significant growth suppression and apoptotic tumor cell death in prostate cancer in vivo.

To accomplish efficient nonviral gene therapy against prostate cancer (PC), Epstein-Barr virus (EBV)-based plasmid vectors containing EBNA1 gene and oriP were employed and combined with a cationic polymer or cationic lipid. When EBV-plasmid/poly-amidoamine dendrimer complex was injected into PC-3-derived tumors established in severe combined immunodeficiency mice, a considerable expression of marker gene was obtained in the tumors, and the expression level was more than eight-fold higher than that achieved by conventional plasmid vector/dendrimer. Since most PC cells express the apoptotic signal molecule Fas (Apo-1/CD95) on their surface, Fas ligand (FasL) gene was transferred into PC cells to kill the tumor cells. In vitro transfection with pGEG.FasL (an EBV-plasmid with the FasL gene) significantly reduced the viability of PC cells, which subsequently underwent apoptosis. Intratumoral injections of pGEG.FasL into PC induced significant growth suppression of the xenograft tumors, in which typical characteristics of apoptosis were demonstrated by TUNEL staining and electron microscopic observations. When pGEG.FasL transfer was accompanied by systemic administrations of cisplatin, the tumors were inhibited even more remarkably, leading to prolonged survival of the animals. FasL gene transfection by means of EBV-based plasmid/cationic macromolecule complexes may provide a practical therapeutic strategy against PC.

Definitive chemoradiotherapy for patients with malignant stricture due to T3 or T4 squamous cell carcinoma of the oesophagus.

We retrospectively investigated the efficacy and feasibility of concurrent chemoradiotherapy for patients with severe dysphagia caused by oesophageal squamous cell carcinoma. Concurrent chemoradiotherapy was performed in 57 patients with T3 or T4 disease containing M1 lymph node (LYM) disease. Chemotherapy consisted of protracted infusion of 5-fluorouracil (5-FU) 400 mg m(-2) 24 h(-1) on days 1-5 and 8-12, combined with 2-h infusion of cisplatin (CDDP) 40 mg m(-2) on days 1 and 8. Radiation treatment at a dose of 30 Gy in 15 fractions of the mediastinum was administered concomitantly with chemotherapy. A course schedule with 3-week treatment and a 1 to 2-week break was applied twice, with a total radiation dose of 60 Gy, followed by two or more courses of 5-FU and CDDP. In all, 24 patients (42%) achieved a complete response, and the 3-year survival rate was 19%. Major toxicities were leukocytopenia and oesophagitis, and there were two (4%) treatment-related deaths. In contrast, 22 patients with T3 disease survived longer than 35 patients with T4 disease (P=0.001); however, the survival rate in 15 patients with M1 LYM disease did not differ significantly from that in 42 patients without M1 LYM disease (P=0.3545). Our results indicate that definitive chemoradiotherapy is potentially curative for locally advanced oesophageal carcinoma with malignant stricture. The efficacy and survival of patients treated with this regimen are related to the T factor.

Cisplatin-induced in vivo differentiation of human embryonal carcinoma.

OBJECTIVE: To investigate the differentiation of embryonal carcinoma (EC) by cisplatin, and the underlying mechanism, as untreated metastases of nonseminomatous germ cell tumours rarely consist of fully differentiated mature somatic tissues, but such mature metastases are more common after various treatments, including chemotherapy. MATERIALS AND METHODS: The TTSC-3 human testicular EC line heterotransplanted into nude mice was used as a target. After treating tumour-bearing mice with intraperitoneal injections of varying doses of cisplatin, the histopathology of the tumours was assessed and various gene expressions in the tumours determined by cDNA-array technology. RESULTS: When cisplatin at 1 mg/kg/week was injected intraperitoneally into TTSC-3-bearing mice, there was no effect on tumour growth. However, injecting cisplatin at 5 mg/kg/week induced a marked regression of the tumour. In contrast, cisplatin at 3 mg/kg/week had a modest inhibitory effect on tumour growth and induced tumour dormancy. Histological examination showed that 5 weeks after injecting cisplatin (3 mg/kg/week), primitive mesenchymal-like cells increased, and 10 weeks afterward cartilage and well-developed glands (teratoma) were apparent; at > 15 weeks afterward there were no EC cells visible. cDNA probes from reverse-transcribed mRNAs of TTSC-3 treated with cisplatin or saline for 10 weeks were compared to identify genes differentially expressed in cisplatin-treated TTSC-3. Of 1176 different human cDNA transcripts in cisplatin-treated TTSC-3, three genes (tumour necrosis factor receptor 1, caspase 8 and Apaf1), which are associated with apoptosis, were expressed markedly more than after saline injection. CONCLUSIONS: The intermediate dose of cisplatin inhibited tumour growth of EC by inducing differentiation and enhancing apoptosis-related gene expression. These findings suggest that cisplatin may play a significant role in the differentiation of EC in vivo.

Potentiation of the sensitivity of renal cell carcinoma cells to TRAIL-mediated apoptosis by subtoxic concentrations of 5-fluorouracil.

Cytotoxic chemotherapy has shown little antitumour activity against renal cell carcinoma (RCC). Although immunotherapy is relatively effective against RCC, the response rate is approximately 20%. Therefore, there is an urgent need to increase this response rate. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo-2L) is one member of the tumour necrosis factor ligand family that selectively induces apoptosis of cancer cells. Since several cytotoxic anticancer drugs including 5-fluorouracil (5-FU) also mediate apoptosis, we reasoned that combined treatment of cancer cells with TRAIL and drugs might result in synergy and overcome the resistance of the cancer cell. This study has examined whether TRAIL can synergise with 5-FU in both cytotoxic and apoptotic assays against drug-resistant RCC cells. Cytotoxicity was determined by an 1-day microculture tetrazolium dye assay. Synergy was assessed by isobolographic analysis. Treatment of Caki-1 cells with TRAIL in combination with 5-FU resulted in a synergistic cytotoxic effect. Synergy was also achieved in freshly derived RCC cells from 3 patients. The enhanced cytotoxicity was obtained irrespective of the sequence of the treatment, but the highest cytotoxicity was observed when Caki-1 cells were treated with TRAIL and 5-FU simultaneously. The synergy achieved in cytotoxicity with TRAIL and 5-FU was shown to be due to apoptosis. The mechanisms responsible for the synergistic cytotoxicity and apoptosis were examined. Treatment of Caki-1 cells with 5-FU enhanced the expression of p53 and bax, but had no effect on the expression of bcl-2. Incubation of Caki-1 cells with TRAIL enhanced the intracellular accumulation of 5-FU and 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP). Treatment of Caki-1 cells with TRAIL downregulated the expression of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) modestly, and upregulated the expression of orotate phosphoribosyltransferase (OPRT). However, the expression level of thymidine phosphorylase (TP) was not affected by TRAIL. This study demonstrates that combined treatment of RCC cells with TRAIL and 5-FU overcomes their resistance. The sensitisation obtained with freshly isolated RCC cells required low subtoxic concentrations of 5-FU. These findings support the potential application in vivo of a combination of TRAIL and 5-FU in the treatment of TRAIL/5-FU-resistant RCC.

Nuclear localization of neutral sphingomyelinase 1: biochemical and immunocytochemical analyses.

To examine the intracellular localization of neutral sphingomyelinase 1 (nSMase 1), a rabbit polyclonal antibody was raised against a recombinant form of the enzyme expressed in E. coli. It has been reported that, in rat liver or in ascites hepatoma AH7974, high activity of neutral sphingomyelinase (SMase) is found at the plasma membrane, with a lesser but significant amount in nucleus and cytoplasm. The biochemical properties, dithiothreitol requirement and high salt concentration dependency, of cloned and expressed nSMase 1 resemble those of previously described nuclear neutral SMase of AH7974. The present study was therefore focused on the nuclear localization of this enzyme. Western blotting of subcellular fractions using anti-rat nSMase 1 antibody revealed most nSMase 1 to be associated with the nuclei and some with microsomes, but not with plasma membranes. Consistently, neutral SMase activity in nuclear extract was immunoprecipitated by the antibody, while that of plasma membranes was not. The results indicate that nSMase 1 mainly resides in the nucleus and may thus differ from neutral SMase in plasma membrane. On gel-filtration column chromatography of nuclear extract, the profile of neutral SMase activity corresponded well with immunoreactive protein bands on western blotting, suggesting that a large part of nuclear neutral SMase may be nSMase 1. Removal of the nuclear envelope by treatment with Triton X-100 did not significantly decrease the amount of nuclear nSMase 1, and western blotting of subnuclear fractions (i.e. nuclear envelope, chromatin, and nuclear matrix) revealed nSMase 1 signal exclusively in the nuclear matrix. Immunocytochemistry with AH7974, as well as rat fibroblast cell line 3Y1, demonstrated nSMase 1 to be localized mainly in the nucleus, with some in the cytoplasm. Moreover, immuno-electron microscopy clearly showed the signal of nSMase 1 to be more dense in the nucleus than in the cytoplasm of AH7974.