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In the present study, we showed that SPA-1, a Rap1 GTPase-activating protein (GAP), was bound to a cytoskeleton-anchoring protein AF-6. SPA-1 and AF-6 were co-immunoprecipitated in the 293T cells transfected with both cDNAs as well as in normal thymocytes. In vitro binding studies using truncated fragments and their mutants suggested that SPA-1 was bound to the PDZ domain of AF-6 via probable internal PDZ ligand motif within the GAP-related domain. The motif was conserved among Rap1 GAPs, and it was shown that rapGAP I was bound to AF-6 comparably with SPA-1. RapV12 was also bound to AF-6 via the N-terminal domain, and SPA-1 and RapV12 were co-immunoprecipitated only in the presence of AF-6, indicating that they could be brought into close proximity via AF-6 in cells. Immunostaining analysis revealed that SPA-1 and RapV12 were co-localized with AF-6 at the cell attachment sites. In HeLa cells expressing SPA-1 in a tetracycline-regulatory manner, expression of AF-6 inhibited endogenous Rap1GTP and beta(1) integrin-mediated cell adhesion to fibronectin in SPA-1-induced conditions, whereas it affected neither of them in SPA-1-repressed conditions. These results suggested that AF-6 could control integrin-mediated cell adhesion by regulating Rap1 activation through the recruitment of both SPA-1 and Rap1GTP via distinct domains.

Purpose Surgeons are increasingly being faced with the problem of treating elderly colon cancer patients. The purpose of this study was to elucidate the feasibility of single-incision laparoscopic surgery for these patients. Methods Among 34 right colon cancer patients treated with single-incision laparoscopic surgery procedure between August 2010 and September 2011, 9 (26.5%) were aged 80 or over. The results of treatment in this elderly group were compared retrospectively with those in 10 younger colon cancer patients (age, 59 to 67 y; control group, 29.5%). Results The sex distribution, body mass index, and the tumor location were similar between the groups. The elderly had a higher incidence of preoperative risk factors (77.7% vs. 40.0%; P=0.17). However, operative time and estimated blood loss were similar and postoperative complications had not occurred in both groups. Conclusions We believe that single-incision laparoscopic colectomy can be carried out safely in elderly patients with colon cancer.

Synchronous colorectal tumors that require surgical treatments are rare. Preliminary experience with concurrent single-incision laparoscopic right hemicolectomy and sigmoidectomy for synchronous carcinoma is reported. A 61-year-old woman presented to our department for the close examination of a bloody stool. Colonoscopy revealed two masses in the right-sided transverse colon and sigmoid colon and another slightly elevated lesion in the transverse colon, and all biopsies from these three lesions demonstrated adenocarcinoma. Under the diagnosis of transverse colon cancers and sigmoid colon cancer, we performed simultaneous single-incision laparoscopic sigmoidectomy and right hemicolectomy. First, a lap protector was inserted through a 2.5 cm transumbilical incision. Three 5 mm ports were placed in the lap protector. We successfully performed sigmoidectomy and right hemicolectomy with lymph node dissection. The patient was discharged on the thirteenth postoperative day. Postoperative follow-up did not reveal any umbilical wound complications. SILS should be the treatment of choice for concurrent laparoscopic surgery for also the other diseases.

Mucocele of the appendix is a rare lesion which denotes a distension of the lumen due to accumulation of mucoid substance, and a possible rupture of the mucocele may result in the clinical condition of pseudomyxoma peritonei. Therefore, the laparoscopic approach for this disease is still controversial because improper handling may cause inadvertent rupture. We present a successfully treated case with the single-incision laparoscopic approach. An 88-year-old man was diagnosed with the mucocele of the appendix, and we decided to perform the single incision laparoscopic ileocecal resection. First, a lap protector was inserted through a 3.0 cm transumbilical incision. Three 5 mm ports were placed in EZ access mounted on the lap protector. On the observation of laparoscopy, a retrocecal appendix was found. We successfully mobilized the right colon and transected the ileum using an endoscopic linear stapler. The right colon with appendix was carefully extracted through the umbilical incision. Resection and anastomosis were achieved following extracorporealization. The appendix was measured 7 cm in length and 3 cm in diameter; a final histopathological diagnosis was mucocele caused by mucinous cystadenoma. This is, to our knowledge, the first case of a single-incision laparoscopic ileocecal resection for mucocele of the appendix described in the indexed literature.

Activation of T cells by antigen requires adhesive interactions with antigen-presenting cells (APC) in which leukocyte function-associated antigen 1 (LFA-1) and intercellular adhesion molecules (ICAMs) are important. However, it is not well understood what signaling molecules regulate this process and how the modulation of adhesive events influences T-cell activation. Here we show that Rap1 is activated in T cells in an antigen-dependent manner and accumulated at the contact site of T-cell and antigen-loaded APC. Inhibition of Rap1 activation by a dominant-negative Rap1 or SPA-1, a Rap1 GTPase-activating protein, abrogates LFA-1-ICAM-1-mediated adhesive interactions with antigen-pulsed APC and the subsequent T-cell-receptor triggering and interleukin-2 production. Conversely, augmented antigen-dependent Rap1 activation by the expression of wild-type Rap1 enhances these responses but culminates in apoptosis by Fas and FasL. Thus, Rap1 functions as a key regulator of T-cell and APC interactions and modulates T-cell responses from productive activation to activation-induced cell death by regulating the strength of adhesive interactions. Moreover, constitutive Rap1 activation rendered T cells unresponsive with accumulation of p27(Kip1). Our study indicates that the activation state of Rap1 has a decisive effect on the T-cell response to antigen.

Among several resistant mechanisms of trastuzumab for HER2+ breast cancer patients, little is clear on resistant mechanism of trastuzumab mediated ADCC. In the current study, we investigated the role of Vacuolar-ATPase subunit ATP6V1B1 on the resistant mechanism of trastuzumab mediated ADCC in HER2+ human breast cancer cell line SK-BR-3. To establish the trastuzumab mediated ADCC resistant SK-BR-3, PBMCs are co-cultured with 1µg/ml of trastuzumab and SK-BR-3 for 4 hours. We removed CD45 positive PBMC by using MACS separation system and obtained ADCC resistant SK-BR-3. After we expanded the resistant cells and repeated these processes several times, we established the ADCC resistant SK-BR-3. The cytotoxicity of ADCC resistant SK-BR-3, which was evaluated by LDH assay, was significantly reduced as compared with that of normal SK-BR-3 (38.7% and 67.4%, respectively). To sort candidate genes for the resistance, we analyzed the gene expression profile of ADCC resistant SK-BR-3 by using DNA microarray and we focused on ATP6V1B1 gene that was significantly reduced on the resistant cells. We investigated ATP6V1B1 mRNA level of SK-BR-3 and resistant cell by using real time PCR and found the 50% reduction of ATP6V1B1 mRNA level in ADCC resistant cells. To evaluate the role of ATP6V1B1 on the trastuzumab mediated ADCC resistant mechanism, we established ATP6V1B1 knockout SKBR3 by using CRISPR/Cas9 system. We cultured from small numbers of the knock out cells and expand to obtain completely knock out cells. We confirmed that the ATP6V1B1 knockout cell line was knocked out by western blotting and immunohistochemistry. We found that ATP6V1B1-knockout SK-BR-3 were significantly less ADCC activity as compared to control SK-BR-3(67.4% and 21.2%, respectively). Furthermore, as the same as the result of ADCC activity, we found that ATP6V1B1-knockout SK-BR-3 was less AICC (antibody-independent cellular cytotoxicity) activity as compared to control SK-BR-3(41.2% and 12.5%, respectively). Based on the in vitro findings, we evaluated the role of ATP6V1B1 expression in HER2 positive breast cancer patients who are treated with neoadjuvant chemotherapy (NAC) containing trastuzumab. Biopsy samples before NAC were stained with ATP6V1B1 immunohistochemically. Four pathological complete response (pCR) cases after NAC and 4 non pCR cases were included. We found that non pCR cases showed significantly week expression of the ATP6V1B1 protein as compared to that of pCR cases. ATP6V1B1 gene is one of the isoforms of the Vacuolar (V)-ATPase which is located in intracellular membrane that regulate the cytosolic pH and membrane trafficking. It is hypothesized that ATP6V1B1 is involved in somewhere through the process of cytotoxicity mediated by perforin and granzymes and now we are trying to explore the mechanisms further. Citation Format: Mariko Nishie, Eiji Suzuki, Kosuke Kawaguchi, Ayane Yamaguchi, Moe Tsuda, Takeshi Kotake, Masahiro Hirata, Fengling Pu, Keiko Sakamoto, Masakazu Hattori, Tomoharu Sugie, Masakazu Toi. Down regulation of ATP6V1B1 in HER2+ breast cancer leads resistance against trastuzumab mediated antibody dependent cellular cytotoxicity and it might be associated with less efficacy of trastuzumab clinically [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4151. doi:10.1158/1538-7445.AM2017-4151

Aim: Single-incision laparoscopic colectomy (SILC) often requires steeper Trendelenburg positioning to displace or keep the small intestine away from the operative site. We have developed hybrid SILC in which we make a transumbilical incision to extract the specimen first and utilize a multiflap gate (MFG). Methods: MFG was inserted through a 4.0-cm transumbilical incision, and a surgical towel was inserted via MFG and displaced the small intestine away from the operative site. Three 5-mm ports were placed in the converter sheet. Almost all the operative procedures were the same as usual laparoscopic sigmoidectomy. In the course of laparoscopic procedures, whenever we felt stress, we used the techniques of open surgery via MFG. Results: In 3 patients, the procedure was successfully completed without any complications. Conclusions: Our procedure can be easily performed, which enables surgeons to achieve SILC safe and easy compared with conventional technique.

There have been significant advances in the treatment of patients with lymphoma and myeloma. Although the improvements in survival outcome have been clearly addressed by clinical trials, these studies includes patients who are otherwise healthy and would be eligible for trials that the actual improvement in survival in the general patient population over time is yet to be elucidated. Therefore, we reviewed the cancer-registry data of patients with lymphoma and myeloma in Japan from 1993 to 2006 and estimated relative survival (adjusted for competing causes of death in same-age members of the general population) according to three periods of diagnosis (1993-1997, 1998-2002 and 2003-2006). We also estimated conditional 5-year relative survival (5-year survival rate of patients who have survived 5 years). A total of 26,141 patients were reviewed and analyzed. Relative survival improved in Hodgkin lymphoma (HL, N = 853, +20% improvement), diffuse large B-cell lymphoma (DLBCL, N = 4,919, +14% improvement) and follicular lymphoma (FL, N = 1,333, +13% improvement). In contrast, we found no significant improvement in survival since 1993 in peripheral T-cell lymphoma (PTCL, N = 667, +4% improvement), adult T-cell leukemia/lymphoma (ATLL, N = 2,166, -5% improvement) or multiple myeloma (MM, N = 4,914, -2% improvement). Conditional 5-year survival of HL, DLBCL, FL, PTCL, ATLL and MM was 88, 87, 79, 63, 53 and 45%, respectively. Relative survival of patients with HL, DLBCL and FL significantly improved from 1993 to 2006 in Japan; in contrast, no improvement was seen in other diseases, suggesting unmet need of novel treatment strategies.

Aim : The safety and efficacy of single-incision laparoscopic resections for patients with stage IV colorectal cancer have not been examined explicitly. This article describes our experience with single-incision laparoscopic procedures for patients with stage IV colorectal cancer. Methods Seventy-seven patients who underwent single-incision laparoscopic colectomy between August 2010 and January 2012 were investigated retrospectively. Eleven patients were in clinical stage IV (ST4 group) and were compared with 66 patients in clinical stages 0 to III (control group). Results There were no differences in the intraoperative and the postoperative complications, the 30-day mortality rate, the number of the lymph nodes harvested, and the duration of postoperative hospital stay between the 2 groups. Conclusions Our initial experiences suggested that single-incision laparoscopic colectomy is feasible for stage IV colon cancer patients. This is a good start comparing the outcomes of single-incision colectomy in stage IV patients with open and traditional laparoscopic colectomy.

Delta-like (dlk) is a family of transmembrane proteins containing epidermal growth factor-like repeat motifs homologous to the notch/delta/serrate family. Recent studies suggest that dlk is a negative regulator of adipocyte differentiation, a promoting factor of cobblestone area colony formation, and a molecule which influences stromal cell-pre-B cell interactions and augments cellularity of developing thymocytes. However, the role of dlk in regulating the growth and differentiation of hematopoietic progenitors remains unclear. In the present study, we examined the effect of dlk on the proliferation of murine hematopoietic progenitors by hematopoietic growth factors. Soluble dlk-IgG Fc chimeric protein completely inhibited the colony formation of lineage-marker negative (Lin-) bone marrow cells by GM-CSF, G-CSF, or macrophage-CSF (M-CSF) in the presence of stem cell factor (SCF). However, dlk failed to inhibit the colony formation of Lin- bone marrow cells by CSF, as described above, or M-CSF plus interleukin 3. Furthermore, dlk failed to inhibit the colony formation of Hes-1-null fetal liver cells by M-CSF in the presence of SCF. These findings suggest that dlk is an important regulator of hematopoietic progenitor proliferation. Depending on the presence of SCF, dlk may act as a growth inhibitor, although dlk signaling does not mediate Hes-1 transcription factor.

Differential expression of renal AGE-receptor genes in NOD mice: Possible role in nonobese diabetic renal disease.BackgroundNonobese diabetic mice (NOD) are prone to glomerular pathology, which is accelerated with the onset of diabetes. Advanced glycation end product (AGE) interactions with AGE-receptors (AGE-Rs) in kidneys can contribute to glomerular injury and diabetic nephropathy (DN). The significant elevation in kidney AGE deposits noted in prediabetic NOD mice suggested that delayed AGE turnover in this model may contribute to its propensity toward DN.MethodsTo explore whether excess tissue AGE was linked to altered AGE-R status in the kidney, mRNA/protein expression, and of several AGE-Rs [AGE-R1, AGE-R2, AGE-R3, scavenger receptor II (ScR-II), and receptor for AGE (RAGE)], was determined in renal cortex and in mesangial cells (MCs) isolated from ND-, D-NOD, and ILE mice (N = 20 per group). Ligand binding, receptor site number, and affinity were determined in MCs from the same mouse groups.ResultsPrediabetic NOD kidney AGE-R1 mRNA and protein level were threefold lower than that of ILE mice (P < 0.01), while AGE-R3 mRNA was enhanced by twofold (P < 0.05) and AGE-R2, RAGE, and ScR-II mRNA remained close to normal (ILE). The onset of diabetes in NOD mice, while enhancing AGE-R1 mRNA expression by approximately twofold, failed to raise it above the normal (ILE) level, despite increases in tissue, and serum AGE. The latter was associated with higher elevation in AGE-R3 (sixfold, P < 0.05), RAGE (twofold, P = NS), and ScR-II mRNA (2.8-fold, P = NS) above control. MCs from prediabetic NOD mice showed a threefold lower level of AGE-R1 mRNA (P < 0.02 vs. ILE) and AGE-R1-protein, and AGE-binding activity (

To identify the intracellular signals which increase the adhesiveness of leukocyte function-associated antigen 1 (LFA-1), we established an assay system for activation-dependent adhesion through LFA-1/intercellular adhesion molecule 1 ICAM-1 using mouse lymphoid cells reconstituted with human LFA-1 and then introduced constitutively active forms of signaling molecules. We found that the phorbol myristate acetate (PMA)-responsive protein kinase C (PKC) isotypes (alpha, betaI, betaII, and delta) or phosphatidylinositol-3-OH kinase (PI 3-kinase) itself activated LFA-1 to bind ICAM-1. H-Ras and Rac activated LFA-1 in a PI 3-kinase-dependent manner, whereas Rho and R-Ras had little effect. Unexpectedly, Rap1 was demonstrated to function as the most potent activator of LFA-1. Distinct from H-Ras and Rac, Rap1 increased the adhesiveness independently of PI 3-kinase, indicating that Rap1 is a novel activation signal for the integrins. Rap1 induced changes in the conformation and affinity of LFA-1 and, interestingly, caused marked LFA-1/ICAM-1-mediated cell aggregation. Furthermore, a dominant negative form of Rap1 (Rap1N17) inhibited T-cell receptor-mediated LFA-1 activation in Jurkat T cells and LFA-1/ICAM-1-dependent cell aggregation upon differentiation of HL-60 cells into macrophages, suggesting that Rap1 is critically involved in physiological processes. These unique functions of Rap1 in controlling cellular adhesion through LFA-1 suggest a pivotal role as an immunological regulator.

Nonobese diabetic (NOD) mice develop glomerulosclerosis shortly after the onset of diabetes. We showed that mesangial cells (MCs) from diabetic mice exhibited a stable phenotypic switch, consisting of both increased IGF-1 synthesis and proliferation (Elliot SJ, Striker LJ, Hattori M, Yang CW, He CJ, Peten EP, Striker GE: Mesangial cells from diabetic NOD mice constitutively secrete increased amounts of insulin-like growth factor-I. Endocrinology 133:1783-1788, 1993). Because the extracellular matrix (ECM) accumulation in diabetic glomerulosclerosis may be partly due to decreased degradation, we examined the effect of excess IGF-1 on collagen turnover and the activity of metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) in diabetic and nondiabetic NOD-MC. Total collagen degradation was reduced by 58 +/- 18% in diabetic NOD-MCs, which correlated with a constitutive decrease in MMP-2 activity and mRNA levels, and nearly undetectable MMP-9 activity and mRNA. TIMP levels were slightly decreased in diabetic NOD-MC. The addition of recombinant IGF-1 to nondiabetic NOD-MC resulted in a decrease in MMP-2 and TIMP activity. Furthermore, treatment of diabetic NOD-MC with a neutralizing antibody against IGF-1 increased the latent form, and restored the active form, of MMP-2. In conclusion, the excessive production of IGF-1 contributes to the altered ECM turnover in diabetic NOD-MC, largely through a reduction of MMP-2 activity. These data suggest that IGF-1 could be a major contributor to the development of diabetic glomerulosclerosis.

Mouse Spa-1 gene with a region homologous to the human rap1GAP gene is transcriptionally induced in the lymphocytes by mitogenic stimulation. Herein we have cloned a cDNA for its human counterpart. SPA-1 cDNA encodes a 130-kDa protein (p130(SPA-1)) consisting of proline-rich regions and rap1GAP-related domain followed by a coiled-coil stretch. Baculovirally expressed p130(SPA-1) exhibited GTPase-activating protein (GAP) activity for Rap1 and Rap2, but not for Ras, Rho, Cdc42, Rac, and Ran, with comparable specific activity to the rap1GAP gene product (p85/95(rap1GAP)). In the cells, p130(SPA-1) was mostly localized at the perinuclear membranous region co-localizing with Rap1 and Rap2. Expression of SPA-1 and rap1GAP genes tended to be segregate in various tissues, lymphoid tissues expressing abundant SPA-1 transcript without rap1GAP, while those such as brain, kidney, and pancreas exhibiting rap1GAP mRNA with little SPA-1. Promyelocytic HL-60 cells, which expressed p130(SPA-1) with little p85/95(rap1GAP) in uninduced state, showed progressive decline in p130(SPA-1) and conversely drastic increase in p85/95(rap1GAP) as they ceased from proliferation and differentiated into macrophages by 12-O-tetradecanoylphorbol-13-acetate. These results suggested that products of SPA-1 and rap1GAP genes, albeit comparable GAP activity for Rap1 and Rap2, functioned in the distinct contexts depending on cell types and/or states.

A 50-year-old woman was seen at the hospital because of hematemesis and tarry stool. Emergency endscopic examination revealed a submucosal tumor with central easy bleeding ulcer at the anal side of the Papilla Vateri of the duodenum. Preoperative examination showed that the tumor which was 5cm in diameter, solid, well-delineated, and hypervascular, developed extramurally from the second portion of the duodenum to the head of pancreas. It was diagnosed as leiomyoma or leiomyosarcoma and a pancreatoduodenectomy was performed. Histopathologically, this tumor was shown to be composed of proliferated spindle cells which arranged in fascicles and were complicated. Amyloid protein deposition was recognized in this tumor. Immunohistochemical study showed that the tumor was negative for α-smooth muscle actin, desmin, S-100 protein and NSE, but was positive for vimentin only. Therefore, it was diagnosed as duodenal stromal tumor whose histogenesis was neither leiomyogenic nor neurogenic. Although mitosis was not seen so frequently in this low cellularity tumor, it was supposed to be possibly malignant because the diameter of this tumor was 5.5cm. We would have to follow up the patient carefully.

Aims/Introduction Despite the use of intensive therapies, declining renal function is often observed during the overt nephropathy stage of type 2 diabetes. We aimed at investigating the role of serum uric acid (SUA) levels at the onset of overt nephropathy in the risk of renal function decline in type 2 diabetes patients. Materials and Methods The present cohort study included 290 type 2 diabetes patients who were followed from the onset of overt nephropathy. The relationship between SUA and declining renal function was assessed using Cox regression models after adjusting for known risk factors. Results Over a median 4.8-year follow-up period, 85 patients (4.9/100 person-years) showed serum creatinine (Cr) doubling with a total cumulative incidence of 71.9% at 20 years of follow up. The highest SUA tertile resulted in significantly a higher incidence (7.7/100 person-years) and cumulative incidence at 20 years (85.7%) than the middle (3.9/100 person-years, 54.2%) and lowest (3.0/100 person-years, 55.5%) tertiles. The univariate Cox hazard model resulted in significant risks for Cr doubling related to female sex, short diabetes duration, smoking and elevated levels of low-density lipoprotein cholesterol (LDL-c), glycated hemoglobin and SUA tertiles. SUA tertiles remained statistically significant in the multivariate model (highest vs lowest hazard ratio 2.68, 95% confidence interval 1.48−5.00, P = 0.0009). Conclusions Elevated SUA levels within the normal range (men >6.3 mg/dL, women >5.1) at the onset of overt nephropathy resulted in an increased risk for declining renal function in type 2 diabetes patients.

OBJECTIVE In Japan, population-based cancer incidence data are reported several years behind the latest year of cancer mortality data. To bridge this gap, we aimed to determine a short-term projection method for cancer incidence. METHODS Data between 1985 and 2007 were obtained from the population-based cancer registries in four prefectures (Miyagi, Yamagata, Fukui and Nagasaki). Three projection models were examined: generalized linear model with age and period (A + P linear); generalized linear model with age, period and their interactions (A*P linear); and generalized additive model with age, period and their interactions smoothed by spline (A*P spline). We performed a 5-year projection for the years 2000 and 2005, based on the data of 1985-95 and 1985-2000, respectively. Seven cancer sites (stomach, liver, colorectal, lung, female breast, cervix uteri and prostate) and all cancers combined were analyzed. The accuracy of projection was evaluated by whether each observed number fell within the 95% confidence interval of the projected number. RESULTS The A*P spline model accurately projected 8 of 13 cancer site-sex combinations, whereas the number of site-sex combinations of accurate projection was 2 and 6 for A + P linear and A*P linear models, respectively. For liver and colorectal cancers, the A*P spline model alone performed accurate projections; the relative differences between projected and observed numbers of cancer incidence ranged between -0.4 and +10.9% for the A*P spline, and between +7.4 and +37.6% for the other two models. All three models failed to project sudden increases in prostate cancer between 2000 and 2005. CONCLUSIONS The A*P spline model is a candidate method for the projection of cancer incidence in Japan. However, we need a continuous validation for prostate cancer.

We report our experience with a case of colorectal cancer treated with chemotherapy for a liver metastasis patient on hemodialysis. The patient was a 67-year-old man with a history of chronic renal failure, who was on hemodialysis since 2005. High anterior resection was performed for sigmoid colon and rectal cancer in January, 2010. After starting chemotherapy while planning to use FOLFOX6+bevacizumab(BV)as a postoperative standard chemotherapy, in combination with hemodialysis three times a week while performing dose escalation, administration postponement was continued for myelosuppression that was considered to be the effect of oxaliplatin. Oxaliplatin was administered for only 2 courses, and was then changed to BV+sLV5FU2 therapy. We continued treating the metastases approximately on schedule. Imaging revealed, the liver metastases were CR because they had disappeared. The BV use case of the dialysis case had few reports, but was thought to be able to use it by careful administration safely.

Our aim was to evaluate postoperative adjuvant chemotherapy using S-1 plus cisplatin(S-1/CDDP)for type 4 gastric cancer.We investigated 18 patients who had undergone curative operations for type 4 gastric cancer. They were classified into two groups of patients, one using S-1/CDDP(group A: 9)and one using S-1 alone(group B: 9), after surgery between 2000 and 2010. Median survival time(MST)and survival rates were reported retrospectively. Patients as- signed to group A were treated with the following regimen: S-1, 80-120mg/day(body surface area 1. 25m2: 80mg/day, 1. 25-1. 5m2: 100mg/day, 1. 5m2: 120mg), was administered for 21 consecutive days followed by a 14-day rest period, and CDDP, 60mg/m2, was administered on day 8 for 5 courses. After this course, S-1 80mg/m2 was given for 18 months. S- 1(80-120mg/day, body surface area 1. 25m2: 80mg/day, 1. 25-1. 5m2: 100mg/day, 1. 5m2: 120mg)was administered for 28 days followed by 14-day rest as one course.MST differed significantly between group A and group B (MST; group A: 1, 603 vs group B: 955 days). The overall survival rate at 5 years was 64. 8% in group A and 13% in group B, and the overall survival rate in group A was statistically better than that in group B(p=0. 02).Postoperative adjuvant chemotherapy using S-1/CDDP for resected type 4 gastric cancer contributes to prolonged life, compared with using S-1 in overall survival.

Marek's disease virus (MDV)-infected chickens show thymic atrophy during the acute phase of infection. We examined whether the thymic atrophy by MDV-infection was mediated by apoptosis. Apoptosis-specific DNA ladderings were clearly observed in thymocytes one week after MDV-infection. Histological and flow cytometry studies revealed that immature CD4+ CD8+ thymocytes underwent apototic cell death. In addition, the expression level of CD8 molecules on both CD4(-)CD8+ and CD4+ CD8+ thymocyte populations was down-regulated in the infected chickens. These thymic changes might be involved in the pathogenesis of Marek's disease.

The function of CD4+ T cells in antibody production was examined by using T cell subset-depleted chickens. CD4- and CD8-depleted chickens, established by the combination of thymectomy and injection of T cell subset-specific monoclonal antibodies, were immunized with sheep red blood cells (SRBC). Titers of anti-SRBC antibody produced in CD4-depleted chickens were lower than those in control chickens, while no difference in the antibody production was observed between CD8-depleted and control chickens. In chickens depleted of both CD4+ and CD8+ T cells, the recovery of T cells in the periphery was demonstrated starting 3 weeks after T cell depletion. Those T cells recovered in the periphery predominantly expressed CD4 molecules. Although low titers of antibody against SRBC were detected in chickens depleted of both CD4+ and CD8+ T cells, an increase of anti-SRBC antibody production was coincidentally observed with the recovery of CD4+ T cells in the periphery. These results suggest that CD4+ T cells could differentiate in extrathymic environments in chickens, and have a helper function in antibody production similar to that of intrathymic T cells. These extrathymic T cells, however, showed a lower proliferative response to concanavalin A than intrathymic T cells, suggesting that these extrathymic T cells may have some properties distinct from intrathymic T cells.

The primary modalities for management of liver abscesses are usually antibiotics and percutaneous drainage. However, in patients with ascites or bleeding tendency, the percutaneous puncture of liver abscesses may be unsuitable. We applied a new approach, nasobiliary tube drainage, for a giant pyogenic liver abscess following diagnostic endoscopic retrograde cholangiopancreatography. Pyogenic liver abscess is often biliary in origin, and this new approach includes assessment of biliary abnormality for the management of the abscess, enabling treatment of parients in whom puncture of the abscess is considered dangerous because of massive ascites around the liver. We propose that this procedure is useful in the management of a subgroup of patients with pyogenic liver abscess. To our knowledge, no previous reports of endoscopic transpapillary abscess drainage in pyogenic liver abscess are available.

Marek's disease virus (MDV) causes T cell immunosuppression in chickens during latent infection. Morphological changes specific to apoptosis were demonstrated in peripheral blood mononuclear cells (PBMC) of MDV-infected chickens at 2-3 weeks post-inoculation (p.i.). Analysis of DNA fragmentation in T cell subsets in the peripheral blood revealed that CD4+ T cells but not CD8+ T cells underwent apoptosis after MDV infection. The proportion of CD4+ T cells, but not that of CD8+ T cells, in the peripheral blood expanded transiently at 16 days p.i., and rapidly decreased 1 week later. The decrease in CD4+ T cells might be mediated by apoptosis, because a rapid reduction in CD4+ T cells was observed when these cells underwent apoptosis. Analysis of the T cell-receptor (TCR) repertoire of the peripheral blood showed that V beta 1 but not V beta 2-alpha beta TCR-bearing cells expanded at 16 days p.i., when the transient expansion of the CD4+ T cell population was observed in these chickens. Flow cytometric profiles also showed that the expression of CD8 was down-regulated after infection with MDV, but there was no difference in the expression level of CD4 molecules between normal and infected chickens. Northern blot analysis indicated that the down-regulation of CD8 occurred at the transcriptional level. These results suggest that both apoptosis of CD4+ T cells and down-regulation of CD8 molecules could contribute to the immunosuppression caused by MDV.

The crystal structures of the hexaphenylditin (hpdt) solvate compounds, (C6H5)3Sn-Sn (C6H5)3 • 2X, solvent X = aniline (an), chlorobenzene (cb), fluorobenzene (fb), and toluene (to), were determined. They are isomorphous with the known benzene (be) crystal solvate compound hpdt • 2be, crystallizing in the trigonal space group R3̅, with Z=3 formula units per unit cell. The lattice constants (in pm), from X-ray powder diffraction, are: hpdt • 2an (1): a= 1170.01 (9), c = 2641.49 (20), c/a = 2.2577; hpdt • 2be (2): a = 1165.45 (5), c = 2641.30 (9), c/a = 2.2663; hpdt • 2cb (3): 0=1175.88(5), c = 2661.66(10), c/a = 2.2635; hpdt • 2fb (4): 0 = 1167.69(5), c = 2643.21 (9), c/a = 2.2636; hpdt • 2to (5): a=1182.24 (7), c = 2649.13(11), c/a = 2.2408. The single crystal structure determination of 5 leads to a = 1180.2(2), c = 2651.4 (5). The decomposition of 5 results in the monoclinic phase of hexaphenylditin. Vapor pressure measurements p=ƒ(T), 260 v/kJmole-1 were determined: 52.44 (1), 46.65 (2), 34.52 (3), 43.08 (4), 55.30 (5). The dynamics of the host molecules C6D6, C6D5CD3 and C6H5ND2 were studied by 2H NMR in the range 295

Previous studies detected a single amino acid substitution (Ala196 to Gly196) between cDNA clones encoding a 32 kDa antigen (p32) of Theileria sergenti (Chitose stock) obtained from a persistently infected calf. In this study, 2 different recombinant baculoviruses ( pAc p32-Ala 196 and pAc p32-Gly 196 ) were constructed for the expression of p32. Molecular masses of the polypeptides produced in Spodoptera frugiperda cells infected with the recombinant baculoviruses were the same as that of antheutic p32. pAc p32-Ala 196 produced additional polypeptides, with molecular masses higher than 32 kDa, which resulted from differential N-glycosylation as revealed by endo N-glycosidase treatment. The results indicate that a single amino acid substitution may lead to a conformational change in p32 which affected post-translational modification of recombinant products.

Although colorectal cancer (CRC), a major cancer worldwide, has shown a proximal or right-sided shift in subsite distribution in western countries, trends in subsite incidence in Asian countries remain unclear. Here, we evaluated subsite-specific trends in CRC incidence rate between 1978 and 2004 in Japan using large data from 10 population-based cancer registries. The colorectal sites (C18-20) were categorized into three groups, proximal colon (C18.0-18.5), distal colon (C18.6-C18.7) and rectum (C19.9 and C20.9). Trends in age-standardized incidence rates (ASRs) were characterized by Joinpoint regression analysis. A total of 474,651 colorectal cancer cases were analyzed. Overall, ASRs increased remarkably until 1993, with an annual percent change (APC) of 4.9%, and then stabilized thereafter. By subsite, however, ASRs of proximal colon significantly increased, with APCs of 7.1% (1978∼1991), 3.8% (1991∼96) and 0.9% (1996∼2004); distal colon showed an initial significant increase, with an APC of 7.6%, but stabilized from 1991 until the end of observation; and rectal cancer showed an initial significant increase, with APCs of 1.9% (1978∼88) and 5.6% (1988∼92), but then decreased abruptly in 1992, the year colorectal cancer screening was introduced nationwide, with an APC of -1.0%. Thus, we revealed that changes in incidence trends for the three anatomic sites apparently began to differ in the 1990s, and this is a first report to our knowledge. We also discuss factors that could explain these changes in Japanese. Careful monitoring is necessary to confirm whether these trends are changing in the Japanese population. Citation Format: Hiroko Nakagawa, Hidemi Ito, Satoyo Hosono, Isao Oze, Haruo Mikami, Masakazu Hattori, Yoshikazu Nishino, Hiromi Sugiyama, Kayo Nakata, Hideo Tanaka. Changes in trends in colorectal cancer incidence rate by anatomic site between 1978 and 2004 in Japan. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5205.

The incidence of cancer of the corpus uteri (CC) in Japan is continuously increasing. To explore the trend in a survival of Japanese CC, we analyzed the population-based cancer registry data. We obtained data on 8,562 CC cases from six prefectural cancer registries. We defined two periods as 1993-2001 (1st period) and 2002-2006 (2nd period). Relative survival (RS) of each period was calculated using cohort analysis, and we compared them using excess mortality model adjusted for age categories and extent of disease. We also estimated RS by age group, extent of disease and histological subtype. For age groups, patients were classified into those aged 15-54, 55-69 and 70-99. For extent of disease, patients were classified into those staged localized, regional and distant group. For histological subgroups, patients were divided into eight histological categories according to ‘Cancer Incidence in Five Continents Vol.10’ partially modified. The 5-year RS was 78.1% in the 1st period and 80.1% in the 2nd period. Compared with the RS in the 1st period, RS in the 2nd period was significantly high [Excess Hazard Ratio (EHR): 0.805, 95%Confidence Interval (95%CI): 0.723-0.896, P In conclusion, we could reveal the improved prognosis of Japanese CC from the period of 1993-2001 to the period of 2002-2006, using population-based cancer registry data. The improvement of short-term RS was observed among elderly, distant or non-endometriod cases, whereas their long-term prognosis did not change during the study period. Improved prognosis might be attributed to the progress of treatment, especially due to chemotherapy or formulation of treatment guidelines in early 2000’s. Citation Format: Shusaku Inoue, Satoyo Hosono, Hidemi Ito, Isao Oze, Yoshikazu Nishino, Masakazu Hattori, Akiko Ioka, Tomio Nakayama, Keitato Matsuo, Mika Mizuno, Kiyoko Kato, Hideo Tanaka, Yuri Ito. Trends in a survival of cancer of the coupus uteri in Japan 1993-2006 (J-CANSIS). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5209.

In this report, we propose a direct patterning method of graphene on the SiC(0001) surface by KrF-excimer-laser irradiation. In this method, Si atoms are locally sublimated from the SiC surface in the laser-irradiated area, and direct graphene growth is induced by the rearrangement of surplus carbon on the SiC surface. Using Raman microscopy, we demonstrated the formation of graphene by laser irradiation and observed the growth process by transmission electron microscopy and conductive atomic force microscopy. When SiC was irradiated by 5000 shots of the laser beam with a fluence of 1.2 J/cm2, two layers of graphene were synthesized on the SiC(0001) surface. The number of graphene layers increased from 2 to 5–7 with an increase in the number of laser shots. Based on the results of conductive-atomic force microscopy measurements, we conclude that graphene formation was initiated from the step area, after which the graphene grew towards the terrace area by further Si evaporation and C recombination with increasing laser irradiation.

The very late antigen-4 (VLA-4) integrin expressed on the surface of lymphocytes and macrophages can regulate their migration to inflammatory sites as well as control cellular activation. The role of VLA-4 in the establishment of autoimmune diabetes is not easily predicted given the multiplicity of adhesion pathways and their differential use by various cell types. The contribution of VLA-4 to insulin-dependent diabetes mellitus was investigated by administration of VLA-4-specific monoclonal antibodies (MoAb) in an adoptive transfer model of disease in NOD mice. This study shows that VLA-4-specific MoAbs profoundly inhibit the development of diabetes with protection sustained by repeated MoAb exposure. Insulitis was completely inhibited during treatment and progressed to a severe degree once MoAb treatment was suspended, yet ∼40% of treated recipients failed to become diabetic during 1–2 months post-treatment. Although we cannot rule out depletion of a relatively minor subpopulation of cells by prolonged anti-VLA-4 MoAb exposure, this inhibition of diabetes onset by treatment with MoAbs to VLA-4 supports a dependence on VLA-4 for cellular functions leading to diabetes and demonstrates that a significant disease modifying effect can be mediated by targeting the VLA-4 integrin.

Temperature dependences of 35Cl and 2H quadrupole coupling constants and 35Cl NMR spin-lat­tice relaxation times in polycrystalline samples were measured in the rotator phase of the butylammonium chlorides C4H9NH3C1 and C4H9ND3C1, obtainable above the phase transition temperature of 241 K. A rapid decrease o f the quadrupole coupling constants of both nuclei upon heating is attributed to increasing dynamic disorder formed around the polar head. The presence of self-diffussion of Cl- ions was revealed from the spin-spin relaxation time and resonance line-width in single crystals, and confirmed by measuring the dc electrical conductivity.

腸管穿通を伴った空腸腸間膜リンパ管腫の1例を経験した. 症例は生後1か月の女児で, 嘔吐, 腹部膨満を主訴に来院腹部の超音波検査, およびCT検査にて, 腸間膜嚢腫によるイレウスと診断, 開腹手術を施行した. Treitz靱帯より約25cmの空腸腸間膜に嚢胞性腫瘤を認め, これにより同部の空腸が圧排・伸展されイレウス状態をきたしていた. この嚢腫を含め小腸を部分切除した. 嚢腫は長径5cm大で多房性であり, 嚢腫に圧排された空腸には打ち抜き様の穿通を認めた. 病理組織学的には海綿状リンパ管腫と診断され, 穿通部腸管の漿膜下組織内にもリンパ管腫が存在した. 腸管壁内のリンパ管腫の存在により, その部のぜい弱化を来し, 腸管が穿通したものと考えられた.小腸腸間膜リンパ管腫は新生児にはまれで, 術前診断は困難なことが多いが, 腹部超音波検査およびCT検査がその診断に有用であった.

T cells of an unique phenotype (CD3+CD4-CD8- alpha beta TCR+) develop in vitro from the hematopoietic progenitors, the majority of which carry homologous alpha beta TCR (V alpha 4J alpha 28/V beta 2D beta 1.1J beta 2.6). In the present study, antigen corresponding to the particular alpha beta TCR was investigated, taking advantage of the fact that growth of T cell hybridomas was arrested by their TCR stimulation. Results indicated that both syngeneic and allogeneic thymocytes, particularly from newborn mice, could specifically inhibit the proliferation of a T cell hybridoma with the V alpha 4/V beta 2 TCR (15H1.2). Proliferation of neither TCR-missing hybridoma subclones nor those with unrelated alpha beta TCRs was affected at all. It was also found that embryonal carcinoma (EC) cells without classical MHC antigens could specifically inhibit the proliferation of 15H1.2 cells. We then raised a mAb, 14.37, against an EC line, OTF9, that could interfere with the ability of them to inhibit the growth of 15H1.2. Pretreatment of 15H1.2 cells with anti-V beta 2 and OTF9 cells with 14.37 mAb respectively completely abrogated the growth inhibition of 15H1.2 by OTF9. The 14.37 antigen was a 120 kDa heterodimer glycoprotein consisting of 85 and 36 kDa proteins. In normal lymphoid tissues, the expression of 14.37 antigen exhibited an apparently inverse relationship with that of class I MHC antigens. Thus, ontogenically it was strongly expressed in fetal and newborn mice, rather rapidly declined thereafter, and remained at very low levels in adult organs. In a given stage, the distribution of 14.37 antigen and class I MHC was rather exclusive to each other.(ABSTRACT TRUNCATED AT 250 WORDS)

A 75-year-old man with type 4 advanced gastric cancer was referred to our hospital. We diagnosed the tumor as cStage III B(cT4a, cN2, cM0)gastric cancer. We selected neoadjuvant S-1 combined with CDDP therapy for him. After 2 courses of chemotherapy, the extension of the gastric wall improved. After an additional 2 courses of chemotherapy, the primary tumor revealed a partial response(PR), judged from a barium meal study and upper GI endoscopic findings, and a total gastrectomy with lymph node dissection was performed. The pathological specimens showed no cancer cells in the gastric wall and lymph nodes, so the histological effect was judged as Grade 3.

A 50-year-old man with advanced gastric cancer and a tumor embolus in the portal vein was referred to our hospital. We diagnosed the tumor as cStage III B (cT3, cN2, cH0, P0, M0) gastric cancer, and selected neoadjuvant S-1 (80 mg/m2) and CDDP (60 mg/m2) therapy for him. After 2 courses of chemotherapy, the embolus in the portal vein disappeared. After additional chemotherapy, the primary tumor and regional lymph node revealed a partial response (PR), and judging from the results from the barium meal study, upper GI endoscopic findings and CT scan, a total gastrectomy with lymph node dissection was performed.

To perform single-incision laparoscopic colectomy (SILC) safely while maintaining the minimal invasiveness of SILC and the quality of the lymph node dissection, we have used hybrid single-incision laparoscopic colectomy (H-SILC). Preliminary experience with H-SILC in advanced colon cancer is reported. First, a multi-flap gate was inserted through a 4.0 cm transumbilical incision, and three 5 mm ports were placed in the converter sheet. The procedures were much the same as in usual laparoscopic colectomy excluding a lateral to medial approach. The initial identification or exposure of the ileocolic vessels was performed through a small incision, and lymphadenectomy was mainly achieved using laparoscopic technique. In the course of laparoscopic procedures, whenever we felt stress, we used the techniques of open surgery through the small incision. The procedure was completed successfully without any perioperative complication and no need to extend the skin incision. The operative time was 191 min. Postoperative follow-up did not reveal any umbilical wound complication or any recurrence. Our experience indicates H-SILC is safe and feasible for selected patients with colon cancer with improved cosmesis.

Malrotation of the midgut is a congenital anomaly of the gastrointestinal tract that usually presents in neonates. Moreover, synchronous colon cancer has rarely been reported. In the present article, we report a preliminary experience with laparoscopic approach for intestinal malrotation with early colon cancer in a 68-year-old woman who presented with bloody stools. Colonoscopy revealed a lateral spreading tumor of the ascending colon. An air-barium contrast enema showed that the entire colon lay within the left hemiabdomen. A computed tomography revealed the superior mesenteric vein rotation sign. At surgery, a condition of malrotation of the midgut was observed: the third and the fourth part of the duodenum descended vertically without Treitz’s ligament, and the small bowel and colon were located in the right and left side of the abdominal cavity, respectively. We mobilized the terminal ileum and the right colon with laparoscopic approach. A 3-cm abdominal incision was made via the umbilicus. Right colectomy with lymph node dissection was achieved following extracorporealization. Pathological examination revealed well-differentiated tubular adenocarcinoma without nodal involvement. The patient had an uneventful postoperative course. Laparoscopic surgery for colon cancer associated with malrotation of the midgut is feasible and a promising method because of its less invasiveness and its adaptability to the malrotation without extending the skin incision.

SUMMARYThe gene for the 32 kDa surface protein (p32) ofTheileria sergentiwas cloned into λgt11 and its nucleotide sequence was determined. The gene encodes a protein of 283 amino acids as deduced from its nucleotide sequence with a 22 residue N-terminal signal peptide. Using this cDNA as a probe we have isolated another two clones from a cDNA library with a CDM8 vector system derived from the same parasite stock. Comparison with three cDNA clones revealed differential polyadenylation and differences in sequences of non-coding regions. Within the coding regions, there were nucleotide transitions which affected thePstI-restriction site, and one of the transitions was also accompanied by an amino acid substitution (Ala to Gly). Southern blot analysis showed hybridization pattern changes among the parasites isolated from individual calves at different times after infection. From these results, we conclude that at least 3 genetically different parasite populations may coexist, and that transition to predominant parasite populations might occur during persistent infections in a host, possibly to evade the host immune responses.

Experimental evidence has suggested that insulin-like growth factor-I (IGF-I) may contribute to diabetic complications. Previously, we and others have shown that normal glomerular mesangial cells have receptors for, synthesize, and exhibit a mitogenic response to IGF-I. We investigated the IGF-I response in cells derived from a genetic model of diabetes, the nonobese diabetic (NOD) mouse. Mesangial cell lines were derived from diabetic (D-NOD) and nondiabetic adult mice. D-NOD cells released more IGF-I into the supernatant and had a decreased binding of IGF-I to surface receptors. Analysis according to Scatchard revealed a decreased number of receptor sites on D-NOD cells, although the structure of the IGF-I receptor visualized by cross-linking was identical for both cell types. Preincubation of D-NOD cells with an antibody to IGF-I resulted in an increase in the number of receptor sites. This suggested that autocrine IGF-I was responsible for the decrease in D-NOD receptor number and that diabetes had resulted in a stable phenotypic change.

Metabolic syndrome and the accompanied diabetes mellitus are both important diseases worldwide due to changes of lifestyle and eating habits. The number of patients with diabetes worldwide is estimated to increase to 300 million by 2025 from 150–220 million in 2010. There are two main types of diabetes. In type 1 diabetes, caused by destruction of pancreatic β-cells resulting in absolute deficiency of intrinsic insulin secretion, the patients require exogenous insulin injections several times a day. In type 2 diabetes, characterized by insulin resistance and abnormal insulin secretion, the patients need exercise, diet control and/or several types of hypoglycemics. The idea of using metal ions for the treatment of diabetes originates from the report in 1899. The research on the role of metal ions that may contribute to the improvement of diabetes began. The orally active metal complexes containing vanadyl (oxidovanadium(IV)) ion and cysteine or other ligands were first proposed in 1990, and a wide class of vanadium, copper and zinc complexes was found to be effective for treating diabetes in experimental animals. We noticed a characteristic compound, allixin, which is a non-sulfur component in dry garlic. Its vanadyl and zinc complexes improved both types of diabetes following oral administration in diabetic animals. We then developed a new zinc complex with thioxoallixin-N-methyl (tanm), which is both a sulfur and N-methyl derivative of allixin, and found that this complex improves not only diabetes but also metabolic syndrome. Furthermore, new zinc complexes inspired from the zinc-tanm were prepared; one of them exceeded the activity of zinc-tanm. The mechanism of such complexes was studied in adipocytes. We describe here the usefulness of the development of metal-based complexes in the context of potential therapeutic application for diabetes and metabolic syndrome.

This study was conducted to clarify the efficacy of centralization of cancer treatment using population-based cancer registry data in Fukui prefecture, Japan.Associations between hospital procedure volume and cancer survival were analyzed using the population-based cancer registry survival data for Fukui prefecture between 1994 and 1998. Firstly the cancer patients who received primary treatments for each target sites such as esophagus, stomach, colon, liver, gall bladder, pancreas, lung, breast, uterus, ovary, prostate, urinary bladder, and lymphoid tissue were totaled. Then, hospitals were divided into 4 categories according to the number of patients by each site; high, medium, low and very low volume. Stage-matched 5-year relative survival rates for each site were then calculated for each categorized hospital volume, and that most desirable for medical treatment for each target site was decided with reference to age-, sex-, and cancer stage-adjusted hazard ratios. Age-adjusted morality reduction was estimated by the expected survival rate after centralization when all cancer patients had received treatments.The 5-year relative survival rates were higher in hospitals with large numbers of patients. With some target sites, such as the stomach, colon, and breast, the mortality was similar between high and low volume hospitals, whereas the other target sites showed higher mortality in line with decrease in number of patients treated. It was estimated that a 2.06% reduction in the mortality rate might be achieved if each case were treated at the most desirable category of hospital in Fukui prefecture.Cancer treatment at hospitals have appropriate procedure volumes is an effective way to increase cancer survival and lower the mortality rate.

We have analyzed the T cell receptor (TcR) V alpha and TcR V beta regions in the spontaneous mouse model for insulin-dependent diabetes mellitus, the NOD mouse, and compared it to the regions in the two sister strains, the NON and CTS strains. Based on restriction fragment length polymorphism analysis the TcR V alpha region in the NOD mouse is essentially identical to that of the SJL/J strain. In contrast both the NON and CTS strains have a unique TcR V alpha haplotype. Whereas the NOD and NON strains apparently contains all the TcR V beta genes, the CTS mouse has three deletions in the V beta region. Our analysis does not give any indications for the diabetic phenotype of the NOD mouse.

Theileria parva is a tick-transmitted intracellular protozoan parasite that causes East Coast fever, a fatal bovine lymphoproliferative disease. The molecular mechanisms that underlie host cell transformation by T. parva schizonts have been studied extensively, and it is known that the nuclear factor-kappa B (NF-kappaB) is activated in schizont-infected cells, making T. parva-transformed cells resistant to apoptosis. However, the mechanism by which the parasite triggers the activation of NF-kappaB remains enigmatic. In the present study, we biochemically characterized a novel protein, which we termed TpSCOP (T. parvaschizont-derived cytoskeleton-binding protein), which is expressed in the schizont stage of T. parva. TpSCOP was shown to interact with F-actin in vitro. Expression of TpSCOP in a murine lymphocytic cell line resulted in the activation of NF-kappaB signaling pathways, leading to apoptosis resistance. The activation of mitogen-activated protein kinase (MAPK), including extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), was also detected. Furthermore, the introduction of TpSCOP into T. parva-infected cells also enhanced the activation of NF-kappaB. This is the first report to demonstrate that a parasite-derived molecule has the ability to activate the host NF-kappaB pathway. Based on these results, TpSCOP likely plays an important role in apoptosis inhibition during Theileria infection.

Cytotoxic T lymphocyte antigen 4 (CTLA-4) is a T cell co-stimulation receptor that delivers inhibitory signals upon activation. This inhibitory effect by CTLA-4 requires activation of small GTPase Rap-1. However, the precise mechanism underlying these negative signals remains unclear. Here, we show that CTLA-4-induced suppression of IL-2 production correlates with rapid destabilization of immunological synapse (IS) formation in murine normal T cell clones. Overexpression of Spa-1, a Rap-1-specific GTPase activating protein (GAP), abolished both Rap-1 activation and IL-2 suppression induced by CTLA-4. Although we failed to find any specific inhibition of activation of early signals upon CTLA-4 engagement, we found that CTLA-4 specifically up-regulates cell motility and suppresses prolonged accumulation of Talin at the contact area with antigen presenting cells upon antigen stimulation. These results suggest that Rap-1 is activated upon CTLA-4 ligation and mediates inhibitory signals through prevention of IS formation.

mGluR6 expression is a characteristic property of retinal ON bipolar cells. mGluR6 is also the causal gene for a form of congenital night blindness. To elucidate physiological and pathological functions of ON bipolar cells and mGluR6, we thought it important to identify genes specifically expressed in them. We thus made transgenic mouse lines expressing humanized Renilla reniformis green fluorescent protein (hrGFP), under the control of the mGluR6 promoter. From their retina, we isolated hrGFP-positive cells by cell sorting, and analysed the gene-expression profile of these cells by using DNA microarray. Further analysis revealed that about half of the initially selected ON bipolar cell genes were expressed in the expected retinal layer. We confirmed previously ambiguous retinal localization of regulator of G-protein signalling 11 (RGS11) and transient receptor potential cation channel, subfamily M, member 1 (TRPM1). In addition, we showed the expression of calcium channel, voltage-dependent, alpha2/delta subunit 3 (Cacna2d3) in ON bipolar cells for the first time. Although we could not completely exclude the possibility that a small population of hrGFP-positive cells might not be ON bipolar cells, these mice as well as our strategy would be highly valuable for the further analysis of ON bipolar cells.

A recombinant baculovirus containing a cDNA which encodes haemagglutinin-neuraminidase (HN) of Newcastle disease virus (NDV) was constructed. Spodoptera frugiperda cells infected with this recombinant virus produced a large amount of HN glycoprotein similar to the authentic HN in size. The recombinant HN glycoprotein was localized on the surface of the infected cells and conserved its haemadsorption and neuraminidase activities. The antigenic properties of the recombinant HN glycoprotein seemed to be slightly different from the authentic one, as judging by the reactivity with a panel of monoclonal antibodies specific to the antigenic sites responsible for neutralization of viral infectivity. Chickens inoculated with the cells infected with the recombinant virus developed haemagglutination-inhibition and virus neutralization antibodies, and were completely protected from the NDV challenge.

A clonal human skeletal muscle cell line showing acid maltase deficiency (AMD) was established through the transfection of origin-defective SV40 DNA. The low acid alpha-glucosidase activity and glycogenosomes in this clone corresponded to AMD. This clone, in spite of loading glycogenososmes, was competent not only as to proliferation without contact inhibition but also as to myogenic differentiation to some extent. Dexamethasone promoted the formation by the transformant of multinucleated myotubes, which expressed acetylcholine receptors. The existence of glycogenosomes did not seem to affect the proliferation or differentiation of myoblasts. The aberrant acid alpha-glucosidase expressed in the transformed myogenic clone was shown to be biochemically identical to that in AMD fibroblasts. This transformant should be of great value for investigating the pathogenesis of AMD because of the possibility of supplying semi-permanently a uniform myogenic cell line expressing AMD.

Although Rap GTPases of the Ras family remained enigmatic for years, extensive studies in this decade have revealed diverse functions of Rap in the control of cell proliferation, differentiation, survival, adhesion, and movement. With the use of genetic engineering strategies, we have uncovered essential roles of Rap signaling in normal lymphohematopoietic cell development as well as its crucial involvement in the development of a wide spectrum of leukemia in manners highly dependent on the contexts of cell lineages. Incidentally, recent results also indicate an important role of Spa-1, a Rap GTPase-activating protein, in invasion and metastasis in human cancers. While it is unlikely that Rap can function as a classic oncogene by itself, like Ras, emerging findings unveil crucial involvements of Rap GTPases in the distinct aspects of malignancy, including leukemia genesis and cancer metastasis. (Cancer Sci 2009; 100: 17–23)