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Background Pulmonary metastasectomy could be considered one of the treatment options for disease control in sarcoma patients with pulmonary metastases; however, there is little consensus regarding the suitable criteria for predicting the likely outcomes in these patients. The aim of this study was to establish a prognostic benefit scoring system based on preoperatively examined prognostic factors for sarcoma patients with pulmonary metastases. Methods This was a single-center, retrospective cohort study conducted in a cohort of 135 sarcoma patients who underwent a first pulmonary metastasectomy at Okayama University Hospital between January 2006 and December 2015. Based on the results of a multivariable logistic regression analysis performed to determine the factors influencing 3-year mortality, a Sarcoma Lung Metastasis Score was created and its correlation with 3-year survival was analyzed. Results The results of the multivariate analysis revealed significant differences in the disease-free interval ( Conclusions Our newly proposed simple Sarcoma Lung Metastasis Score appears to be a useful prognostic predictor for sarcoma patients with pulmonary metastases, in that it could be helpful for the selection of appropriate treatments for these patients.

11576 Background: Malignant soft tissue tumor is a rare cancer with few therapeutic options. Recent genomic analysis of soft tissue sarcoma (STS) revealed a high degree of chromosomal instability (CIN) including genome-wide copy number alteration, aneuploidy, whole genome doubling and chromothripsis. In STS patients, we observed extensive somatic LOH, a hallmark of CIN, which is haploid of germline mutations/variants in cancer-related genes. CIN of STS genome implicates abnormalities in chromosome segregation and mitosis. So far, studies on this issue, however, have not been reported in STS patients. Methods: We recruited 155 patients with metastatic/recurrent malignant soft tissue tumors (135 female and 20 male, mean age 51 at analysis, 100 LMS, 19 LPS, 4 ESS, 3 UPS, 3 AS, 3 MPT, 3 GIST and others) with information of familial cancer burden. Whole exome sequencing and analysis were performed in both blood and tumor samples as described in 2018ASCO. KIF18A expression was assessed by immunohistochemistry. Results: Patients with tumors harboring less than 33% somatic LOH in a total of somatic and LOH mutations/variants in 595 COSMIC genes (n = 54) have a better prognosis than those (34-66%, n = 49, 67-100%, n = 52) with tumors harboring more LOH genes (5-year OS; 71% vs 52% or 46%, p = 0.0299, p = 0.0117, respectively). Neither TMB nor MSI status was associated with LOH. Of the genes involved in chromosome segregation and mitosis, we found that a family of ARHGAP genes which play a role in the spindle assembly and Aurora A kinase activation was frequently mutated in both tumor and germline genomes (n = 81 in a total of 155). Damaging ARHGAP mutations/variants in tumors are correlated with higher LOH values (54±3.4%, n = 81 vs. 39±3.3%, n = 74. mean±SE, p = 0.0021) and poor prognosis of patients (5-year OS; 58% n = 81 vs. 41% n = 74, p = 0.0098). We also found that elevated and robust expression of a mitotic kinesin KIF18A in tumors harboring higher LOH and/or damaging ARHGAP mutations/variants (n = 12) but not in lower LOH tumors without ARHGAP mutations/variants (n = 4). Conclusions: This study, for the first time, demonstrates that genetic abnormalities and aberrant expression of genes involved in chromosome segregation and mitosis in patients with malignant soft tissue tumors. The results reveal a novel target of drug discovery for incurable STS because CIN tumor cells are shown to be particularly vulnerable to KIF18A inhibition, while somatic, diploid cells are not.

To understand the degradation behavior of light water reactor (LWR) fuel-cladding tubes under neutron irradiation, a detailed mechanism of hydrogen pickup related to the point defect formation (i.e., a-component and c-component dislocation loops) and to the dissolution of precipitates must be elucidated. In this study, 3.2 MeV Ni3+ ion irradiation was conducted on Zircaloy-2 samples at room temperature. Thermal desorption spectroscopy is used to evaluate the deuterium desorption with and without Ni3+ ion irradiation. A conventional transmission electron microscope and a spherical aberration-corrected high-resolution analytical electron microscope are used to observe the microstructure. The experimental results indicate that radiation-induced dislocation loops and hydrides form in Zircaloy-2 and act as major trapping sites at lower (400–600 °C) and higher (700–900 °C)-temperature regions, respectively. These results show that the detailed microstructural changes related to the hydrogen pickup at the defect sinks formed by irradiation are necessary for the degradation of LWR fuel-cladding tubes during operation.

To estimate the effect of displacement damage by neutron irradiation of an oxide film of zirconium alloy, a Zircaloy-2 sample was corroded in high temperature and high pressure water at 561 K for 1000 hours to form an oxide film. It was then irradiated with 3-MeV Zr2+ ions up to 1.3 × 1020 ions/m2 at 573 K. Subsequently, the crystalline properties of the oxide film and nature of secondary phase particles (SPPs) in the oxide film were investigated by X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM), and secondary-ion mass spectrometry (SIMS). From the results off the XRD measurement, monoclinic-ZrO2 was found to be predominant at operation temperature of a light-water reactor, and slightly tetragonal ZrO2 was also confirmed. The diffraction peaks of the monoclinic ZrO2 were slightly clearer in the case of the irradiated sample than in the case of the unirradiated sample. Specifically, ratio of tetragonal ZrO2(011) diffraction to monoclinic ZrO2(-111) diffraction was increased by irradaiation. In addition, half width of monoclinic ZrO2(-111) diffraction was decreased with irradiation dose. This result suggests that the crystallinity of the oxide film was macroscopically improved by the ion irradiation. The diffracted wave corresponding to the (011) plane of the tetragonal ZrO2 was also revealed after irradiation. It may suggest that tetragonal ZrO2 is stabilized by accumulation of irradiation defects. However, the crystal structure difference between irradiated and unirradiated is small. Detailed TEM observation of the oxide film confirmed transition from crystalline to amorphous phase in the SPPs near the metal-oxide interface. Furthermore, the diffraction pattern taken from the SPPs near the surface of the oxide film could not be distinguished from that of ZrO2. That is, it was concluded that those near the surface of the oxide film were assimilated into ZrO2. Their iron concentration was less than that near the metal/oxide interface. Following the TEM observation, SEM observation was performed in the vicinity of the area where the TEM image was obtained. According to the SEM observation, the number of SPPs in the ion-irradiated oxide film tended to be lower than that in the unirradiated oxide film. This tendency was more significant near the surface than near the metal-oxide interface. It is therefore concluded that ZrO2 in the oxide film is not easily irradiated; however, the SPPs are affected by the irradiation, and dissolution of the alloying elements from the SPPs to the matrix of the oxide film is promoted by substitution irradiation damage.

Purpose Sarcomas are among the most refractory malignant tumors and often recur as pulmonary metastasis. Although the presence of a high neutrophil-to-lymphocyte ratio (NLR) has been associated with the prognosis of several malignancies, the relationship between the NLR and sarcoma with pulmonary metastasis is unclear. We investigated the impact of the NLR in patients who underwent surgical resection for metastatic lung tumors from various sarcomas. Methods The subjects of this retrospective study were 158 patients with metastatic lung tumors from various sarcomas, who underwent initial pulmonary metastasectomy between 2006 and 2015. We examined the clinicopathological variables, including the NLR and the characteristics of surgical procedures. Survival was estimated by the Kaplan–Meier method and prognostic factors were evaluated by multivariate analysis. Results Multivariate analysis revealed significantly better survival of the group with an NLR 2 years, and 3 or more pulmonary metastasectomies. Conclusion The NLR immediately before the most recent pulmonary metastasectomy is a novel independent prognostic factor, which may be helpful when considering repeated pulmonary metastasectomy.

It is important to clarify the mechanisms of the dislocation loop formation, dissolution of precipitates to understand the degradation behavior of the fuel cladding tubes in light water reactors (L...

11533 Background: Malignant soft tissue tumor is a rare cancer with few therapeutic options. Recent genomic analysis revealed widespread CNA and the cumulative burden of cancer-related pathogenic germline mutations/variants, their clinical and therapeutic significance were unknown. Methods: We recruited 155 patients with advanced malignant soft tissue tumors (135 female and 20 male, mean age 51 at analysis, 100 LMS, 19 LPS, 4 ESS, 3 UPS, 3 AS, 3 MPT, 3 GIST and others) with confirmed metastasis/recurrence and information on familial cancer burden. Whole exome sequencing was performed in both blood and tumor samples as described in 2018ASCO. The copy number of BRCA2 gene was determined by the MLPA method. Tumor immune microenvironment was assessed by immunohistochemistry. The MSI status was analysed by PCR. Results: We analyzed the LOH status in 595 COSMIC genes and found that genome-wide accumulation of somatic LOH of polygenic germline mutations/variants. Patients with more than 33% LOH genes (n=102) in the total of somatic and LOH mutations showed significantly lower OS rates compared with those (n=53) with less LOH genes (5-year survival rates; 49 vs 75%, p=0.010), which constitute 78% of LMS (n=78/100) and 26% of LPS (n=5/19). Total of 41 patients (26%, n=41/155) including 33 LMS (33%, n=33/100) showed LOH in the BRCA2 locus with hemizygous VUS. Those patients with BRCA2 LOH (n=41) showed significantly lower OS rates compared with those without BRCA2 LOH (n=114) (5-year survival rates; 43 vs. 64%, p=0.019). Neither TMB nor the MSI status was associated with LOH. In contrast, accumulation of somatic LOH (mean LOH values of 71.7 vs. 15.7%) was clearly and negatively associated with CD8+T-cell immune infiltrates (T-cells; 44±23 vs 555±180/mm2, n=7, p=0.016), CD20+B-cell accumulation in tertiary lymphoid structures (TLS) (TLS; 0.57±0.43 vs. 20.1±6.1/tumor, n=7, p=0.008) and low levels of neutrophil-to-lymphocyte ratio (NLR) (NLR; 3.63±0.45 vs. 1.71±0.17, n=7, p=0.002), hallmarks of the immunological response to tumors. Conclusions: This study suggests that in advanced malignant soft tissue tumors, accumulation of genome-wide LOH of germline mutations/variants is associated with the BRCAness status and suppression of the immune responses to tumors, and thus influences therapeutic response and survival of the patients.

11531 Background: Malignant soft tissue tumor is a rare cancer with few therapeutic options. Although recent genomic characterization of soft tissue sarcoma revealed massive CNA and an excess of polygenic burden of pathological germline variants, their clinical and therapeutic significance remains to be understood. Methods: We recruited 155 patients with malignant soft tissue tumors (135 female and 20 male, mean age 51, 100 LMS, 19 LPS, 4 ESS, 3 UPS, 3 AS, 3 MPT, 3 GIST and others) of confirmed metastasis/recurrence. Whole exome sequencing was performed as reported in 2018ASCO. The MSI status was analyzed by PCR. Tumor immune microenvironment was assessed by immunohistochemistry. Results: Of the 595 COSMIC genes, heterozygous germline mutations/variants of the genome-wide 0-44 genes (av. 9.7/tumor) showed somatic loss-of-heterozygosity (LOH) with allele frequency of more than 70%. Patients with less than 33% LOH (n=53) in the total of somatic and LOH mutations showed improved 5-year survival rate compared with those (n=102) with more LOH (71% vs 52%, p=0.037). LMS (n=100) had higher value of LOH mutations than other tumors (n=55)(av. 55.5 vs 31.2%, p2). Case 2 had no LOH mutations while case 1 had 37% LOH with more total mutations in tumor (16.1 vs 85.9/Mb). Higher values of LOH (av. 67 vs 19%) were clearly correlated with decreased density of CD8+TIL in tumor tissues (av. 9.6 vs 429/mm2, n=5, p=0.018). Conclusions: Our results, for the first time, suggest that in malignant soft tissue tumors, accumulation of genome-wide LOH of germline mutations/variants, from which self-antigens could be generated, may influence tumor immune microenvironment, and thus influence immunotherapy response and survival of the patients.

Sarcoma is well-known rare cancer with few therapeutic options. Recent comprehensive genomic analyses of adult soft tissue sarcoma revealed few somatic mutations and massive copy number variations(CNVs)by the specific chromosomes. Those features are quite different from the genomics of carcinoma such as lung and colon cancers in which driver and passenger mutations play a central role in the pathogenesis. Furthermore, it has been demonstrated that substantial population of sarcoma patients has pathological germline variants of cancer predisposition genes. These findings imply that, in addition to somatic mutations, inherited germline variants may play a role in the disease state of sarcoma via dosage effects. On this basis, we also discuss on the prospect and limitation of the precision medicine of sarcoma.

Calponin is a family of actin filament-associated regulatory proteins. Among its three isoforms, calponin 1 is smooth muscle specific and calponin 2 is expressed in smooth muscle and certain non-muscle cells. Previous studies showed that calponin 1 knockout mice had detectable changes in the contractility of urogenital smooth muscle whereas other smooth muscles were less affected. To investigate the possibility that calponins 1 and 2 have overlapping functions in smooth muscle, we examined the effect of double knockout of calponin 1 and calponin 2 genes (Cnn1 and Cnn2) on smooth muscle functions. The results showed for the first time that calponin 1 and calponin 2 double knockout in mice does not cause lethality. The double knockout mice showed decreased systemic blood pressure, decreased force development and blunted length tension response in endothelial-removed aortic rings. A compensatory increase of calponin 1 was found in smooth muscle of Cnn2(−/−) mice but not vice versa. Cnn1(−/−) and Cnn2(−/−) double knockout aortic smooth muscle exhibits faster relaxation than that of wild type control. Double deletion or co-suppression of calponin 1 and calponin 2 in vascular smooth muscle to blunt myogenic response may present a novel approach to develop new treatment for hypertension.

11071 Background: Soft tissue sarcoma (STS) is well-known rare cancer with few therapeutic options. Although recent genomic analyses of sarcoma revealed few somatic mutations, massive copy number variations (CNV) and chromoplexy which correlate with worse clinical outcomes, their molecular and genomic mechanisms remain to be understood. Methods: We recruited 116 patients (102 female and 14 male, mean age 50, 80 LMS, 14 LPS, 3 AS and others) and performed whole exome sequencing with the methods as reported in ASCO2018. In addition to somatic mutations, we evaluated germline and CNV contributions in tumor to find LOH mutations by Strelka and Virmid analysis softwares. Results: Of the total of 135-4717 (mean 1129) mutations in tumors, 3-111 (mean 32) mutations were found in 595 COSMIC genes including both somatic and LOH mutations. Less than 33% LOH in the total of somatic and LOH mutations significantly correlated with improved 5-year survival rate as compared with patients with more LOH (81% vs 52%, P=0.01). Among the 224 genes reported in somatic mutations of sarcoma, damaging mutations in ARHGAPs, Rho GTPase signal inactivating genes, were most frequently detected in 59% of total (n=116) and 63% of leiomyosarcoma (n=80) patients as in somatic and/or LOH mutations. Patients with ARHGAP mutations were significantly reduced 5-year survival rates as compared with patients without mutation (51% vs 76%, P=0.007). Among the 163 genes involved in autophagy, a key silencing process for active RhoGTPase, one or more damaging mutations as in somatic and/or LOH mutations were found in 87% in total and 96% in leimyosarcoma patients. Conclusions: Our results, for the first time, suggest an important role of mutations in genes involved in Rho GTPase and autophagy signaling, both well known regulator of chromosomal stability, invasion and metastasis of tumor cells, and thus implicate a potential therapeutic target in STS.

Breakthrough curves of non-trace H 2 –D 2 mixture replacement adsorption with a synthetic zeolite SZ-13X packed column at a cryogenic temperature of 77.4 K were measured at several concentrations of D 2 in H 2 –D 2 mixtures and at the same ones of H 2 in these mixtures, and were analyzed by the curve-fitting with the curves estimated from a model of non-trace two-component mass-flow balances of H 2 and D 2 in the packed column. This dynamic model of adsorption takes in account the characteristics of the separation factor of D 2 /H 2 in adsorption equilibrium depending on the composition in adsorbed phase. Experimental curves show a slight but certain variation with increase in the concentration of a subjective component in the isotope mixtures. It is demonstrated here that these behaviors can be approximated closely by the numerical curves. The curve-fitting analysis brings an interesting result that the overall mass-transfer coefficient of D 2 replacing H 2 adsorbed in advance is larger than that of H 2 replacing D 2 adsorbed. This difference can be interpreted by assuming that desorption of molecules adsorbed in advance is the rate-determining process of replacement adsorption.

Calponin is an actin filament associated regulatory protein in smooth muscle and non-muscle cells. Calponin-1 is specifically expressed in smooth muscle cells and calponin-2 is expressed in both smooth muscle and multiple non-muscle cell types. The role of calponin-1 in the regulation of smooth muscle contraction has been extensively investigated. Calponin-1 gene knockout mice demonstrated minor but clearly detectable changes in smooth muscle contractility, especially in the urogenital system. Here we compared the impacts of single and double knockout of calponin-1 and calponin-2 genes in mice on smooth muscle contractility. The results showed that either calponin-1 or calponin-2 knockout did not significantly alter the passive tension-active tension relationship in aortic smooth muscle and the mice exhibited normal blood pressure. The double knockout mice survived well into adulthood in the absence of physiological stress. However, the double knockout mice showed higher passive tension and less KCl- and norepinephrine-induced active tension in endothelium-removed aorta rings. The double knockout mice also showed lower systolic and diastolic blood pressures compared to gender-matched wild type controls. The results suggest that calponin-1 and −2 both contribute to the regulation of vascular smooth muscle contractility. The minimized phenotypic effects of calponin-1 and calponin-2 single knockout mice indicate a mutual compensation between the two isoforms in smooth muscle cells. Supporting this notion, analysis of protein expression in various types of smooth muscle showed up-regulations of one isoform when the other was deleted. The results implicate that calponins are molecular targets for modifications of smooth muscle contractility and especially blood pressure. Simultaneously targeting the expression and function of both two calponins may provide a potential therapeutic approach to the development of new treatment of hypertension.

Cardiac metastasis of malignant phyllodes tumor is very rare. We herein report a rare case that developed cardiac metastasis from malignant phyllodes tumor. A 38-year-old woman underwent lumpectomy, and the final pathological findings showed the 5-cm malignant phyllodes tumor partially containing 1 cm of squamous cell carcinoma. Four months after the first surgery, a local recurrence of malignant phyllodes tumor and distant metastases to the bone, lung, pulmonary main trunk, and right ventricle were detected. Mass reduction surgery of cardiac metastasis of the malignant phyllodes tumor was performed to avoid sudden death. In immunohistochemical findings, the tumor was suspected to be originated in myoepithelial cells because of the expression of smooth muscle lineage including α-smooth muscle actin and Calponin1 and highly malignant characteristics showing MIB-1 and p53 highly positive with angiogenesis. Further studies are needed to clarify the effective treatment to these tumors.

11571Background: Soft tissue sarcoma (STS) is well-known rare cancer with few therapeutic options. Although recent comprehensive genomic analyses of adult STS revealed few somatic mutations and man...

The functional significance of smooth muscle-specific h1-calponin up-regulation in the smooth muscle contractility of SM-B null mice was studied by generating double knockout mice lacking both h1-calponin and SM-B myosin. The double knockout mice appear healthy, reproduce well and do not show any smooth muscle pathology. Loss of h1-calponin in the SM-B null mice bladder resulted in increased maximal shortening velocity (Vmax) and steady-state force generation. The force dilatation pressure, which was decreased in the SM-B null mesenteric vessels, was restored to wild-type levels in the double knockout vessels. In contrast, the half-time to maximal constriction was significantly increased in the double knockout vessels similar to that of SM-B null mice and indicating decreased shortening velocity in the double knockout vessels. Biochemical analyses showed that there is a significant reduction in smooth muscle α-actin levels, whereas h-caldesmon levels are increased in the double knockout bladder and mesenteric vessels, suggesting that these changes may also partly contribute to the altered contractile function. Taken together, our studies suggest that up-regulation of h1-calponin in the SM-B null mice may be necessary to maintain a reduced level of cross-bridge cycling over time in the absence of SM-B myosin and play an important role in regulating the smooth muscle contraction.

Epidermal growth factor (EGF) and ErbB family molecules play a role in heart development and function. To investigate the role of EGF family member, heparin-binding EGF-like growth factor (HB-EGF) in heart development, smooth muscle and endothelial cell lineage-specific HB-EGF knockout mice were generated using the Cre/loxP system in combination with the SM22alpha or TIE2 promoter. HB-EGF knockout mice displayed enlarged heart valves, and over half of these mice died during the first postnatal week, while survivors showed cardiac hypertrophy. These results suggest that expression of HB-EGF in smooth muscle and/or endothelial cell lineages is essential for proper heart development and function in mice.

Microstructural examination and microhardness test were carried out for unalloyed vanadium, V–5Ti, V–4Cr–4Ti alloys irradiated with 4 MeV Cu ions at temperatures from 200 to 600 � C. In order to investigate the impurity effect, high purification technique was used. Impurity effects can be seen in the nucleation and growth processes of voids in unalloyed vanadium and those of Ti(OCN) precipitates in V–5Ti. The changes in microstructure due to interstitial impurity could not be seen in V–4Cr–4Ti after the irrodiations below 300 � C. The effect of impurity for irradiation hardening in ion-irradiated V–4Cr–4Ti alloys could be seen at 400 � C, but it could not be seen at 200 and 300 � C. On the other hand, dislocation channels were observed at 200 and 300 � C, and the total length of dislocation channels became larger as the impurity level increased. It can be considered that there was practically no correlation between irradiation hardening and total length of dislocation channels due to impurity effects.

We report here mutations affecting various aspects of liver development and function identified by multiple assays in a systematic mutagenesis screen in Medaka. The 22 identified recessive mutations assigned to 19 complementation groups fell into five phenotypic groups. Group 1, showing defective liver morphogenesis, comprises mutations in four genes, which may be involved in the regulation of growth or patterning of the gut endoderm. Group 2 comprises mutations in three genes that affect the laterality of the liver; in kendama mutants of this group, the laterality of the heart and liver is uncoupled and randomized. Group 3 includes mutations in three genes altering bile color, indicative of defects in hemoglobin-bilirubin metabolism and globin synthesis. Group 4 consists of mutations in three genes, characterized by a decrease in the accumulation of fluorescent metabolite of a phospholipase A(2) substrate, PED6, in the gall bladder. Lipid metabolism or the transport of lipid metabolites may be affected by these mutations. Mutations in Groups 3 and 4 may provide animal models for relevant human diseases. Group 5 mutations in six genes affect the formation of endoderm, endodermal rods and hepatic bud from which the liver develops. These Medaka mutations, identified by morphological and metabolite marker screens, should provide clues to understanding molecular mechanisms underlying formation of a functional liver.

A large-scale mutagenesis screen was performed in Medaka to identify genes acting in diverse developmental processes. Mutations were identified in homozygous F3 progeny derived from ENU-treated founder males. In addition to the morphological inspection of live embryos, other approaches were used to detect abnormalities in organogenesis and in specific cellular processes, including germ cell migration, nerve tract formation, sensory organ differentiation and DNA repair. Among 2031 embryonic lethal mutations identified, 312 causing defects in organogenesis were selected for further analyses. From these, 126 mutations were characterized genetically and assigned to 105 genes. The similarity of the development of Medaka and zebrafish facilitated the comparison of mutant phenotypes, which indicated that many mutations in Medaka cause unique phenotypes so far unrecorded in zebrafish. Even when mutations of the two fish species cause a similar phenotype such as one-eyed-pinhead or parachute, more genes were found in Medaka than in zebrafish that produced the same phenotype when mutated. These observations suggest that many Medaka mutants represent new genes and, therefore, are important complements to the collection of zebrafish mutants that have proven so valuable for exploring genomic function in development.

Objectives To investigate the correlation between pathologic findings and maturation of the tumor neovasculature of renal cell carcinoma by immunohistochemical studies. Methods Formalin-fixed and paraffin-embedded specimens from 25 randomly selected patients with renal cell carcinoma were stained with mouse monoclonal antibodies, anti-human CD31, anti-alpha smooth muscle actin (αSMA), and anti-human calponin by the indirect immunoperoxidase method. The microvessels were counted in six areas with the higher number of microvessels in each patient at 200× magnification (0.255 mm 2 per area). Results The number of CD31-positive microvessels in grade 3 tumors was significantly lower than those in grade 1 or 2 tumors ( P = 0.003222 and P = 0.043217, respectively). The CD31-positive microvessel counts of those of higher stage, tumor size greater than 4.5 cm, or non-clear cell type were significantly lower than tumors of lower stage, size less than 4.6 cm, or clear cell type. In the grade 3 tumors, the expression ratio of the number of αSMA-positive microvessels to the number of CD31-positive microvessels was significantly decreased compared with grade 1 or 2 tumors ( P = 0.000011 and P = 0.000000, respectively). The expression of calponin in the tumor neovasculature was not observed. The expression ratios of the number of αSMA-positive microvessels to the number of CD31-positive microvessels in higher stages, larger tumor sizes, or non-clear cell types were significantly decreased. Conclusions The tumor neovasculature of high-grade and high-stage tumors was immature. These results imply that high-grade tumors of renal cell carcinomas may be susceptible to antiangiogenesis therapy inducing apoptosis of immature tumor vessels.

Basic or h1-calponin is a smooth muscle–specific, actin-binding protein that is involved in the regulation of smooth muscle contractile activity. We found in this study the expression of mRNA and protein for h1-calponin in AR42J-B13 cells, which is a useful model for investigating islet β-cell differentiation from pancreatic common precursor cells. Following treatment of AR42J cells with activin A and hepatocyte growth factor, the protein levels of h1-calponin decreased in a time-dependent manner during the course of the cell differentiation. When h1-calponin was continuously overexpressed by utilizing recombinant adenovirus-mediated gene transfer, the percentage of cell differentiation in h1-calponin overexpressing cells was markedly suppressed as compared with that in the cells without overexpression (6.7 ± 2.5 vs. 28.6 ± 3.2%, P < 0.001, Student’s t test). Finally, overexpression of h1-calponin (65.6 ± 3.4), or that lacking actin-binding domain (55.9 ± 3.4%), significantly (P < 0.001) suppressed the activin A–stimulated transcriptional activity of activin responsive element (ARE), whereas calponin homology-domain disruption mutant did not (100.6 ± 1.9%). These results suggest that regulation of h1-calponin is involved in the regulation of differentiation of AR42J cells into insulin-producing cells at least partly through modulating ARE transcriptional activity.

Background/Aims: Malignant fibrous histiocytoma (MFH) of bone, a relatively rare primary malignant bone tumour, is a distinct clinicopathological entity as opposed to MFH derived from soft tissue. Although the true histogenesis of this condition is still controversial, a considerable number of cases of MFH in soft tissue show positive immunohistochemical reactivity for muscle markers such as desmin, common muscle actin (HHF35), and α smooth muscle actin (SMA), suggesting that MFH cells are myofibroblastic in nature. Methods: This study investigated immunoreactivity for several different muscle markers in 19 cases of MFH of bone together with reverse transcription polymerase chain reaction (RT-PCR) analysis on frozen tissue samples that were available in four cases, and compared the data with those found in 11 cases of osteosarcoma and 11 cases of soft tissue MFH treated over the same period. Results: Immunohistochemistry revealed that MFH of bone showed relatively frequent expression of smooth muscle markers, including calponin (nine cases), α-SMA (nine cases), and SM22α (18 cases), and this was confirmed by RT-PCR analysis. However, only one, two, and three cases of MFH of bone showed positive staining for desmin, MyoD1, and HHF35, respectively. Similarly, 11 osteosarcoma cases were relatively frequently positive for α-SMA (five cases), calponin (four cases), and SM22α (seven cases), and less frequently positive for desmin (one case), MyoD1 (none), and HHF35 (none). In contrast, very few MFH of soft tissue cases (n = 11) showed positive reactivity for all of these muscle markers. It has recently been reported that human bone marrow stromal cells also express various kinds of smooth muscle markers including α-SMA and calponin. Conclusions: These results suggested that MFH of bone may derive from mesenchymal stromal cells in bone marrow and has a more myofibroblastic differentiation than soft tissue MFH.

The basic or h1 calponin gene, which encodes an actin-binding protein involved in the regulation of smooth-muscle shortening velocity, is known to be a smooth-muscle differentiation-specific gene. It was found that basic calponin was expressed by cultured mesangial cells and localized along the actin filaments. Among the growth factors involved in the mesangial cell pathophysiology, including platelet-derived growth factor-BB (PDGF-BB), tumor necrosis factor-alpha (TNF-alpha), and transforming growth factor-beta1 (TGF-beta1), TNF-alpha potently downregulates basic calponin expression in both the mRNA and protein levels, whereas TGF-beta1 upregulates the calponin expression. PDGF-BB also reduced its mRNA expression. The half-life of basic calponin mRNA was determined to be similar between TNF-alpha-treated and -untreated mesangial cells, whereas cell transfection assays that used a luciferase reporter gene construct containing the functional basic calponin promoter showed that TNF-alpha and PDGF-BB reduced the transcriptional activity. Because stimulation with TNF-alpha and PDGF-BB was associated with mesangial cell proliferation, basic calponin may play a role in the suppression of mesangial cell proliferation. Treatment with anti-glomerular basement membrane antibody in calponin knockout mice induced more severe nephritis than in wild type mice, as judged from an increase in the urinary protein excretion, glomerular cellularity, and number of proliferating cell nuclear antigen-positive cells in glomerulus. These results suggest that basic calponin expression may serve as one of the intrinsic regulators of glomerular nephritis. Elucidation of the molecular mechanisms for regulation of the basic calponin expression in mesangial cells may improve the understanding of the molecular basis and pathogenesis of the glomerular response to injury.

The mechanical properties of smooth muscles in aorta and vas deferens were studied in mice with a mutated basic calponin locus to learn the physiological function of calponin. The intact smooth muscles were stimulated with high KCl and the force development was compared between calponin deficient (knockout, KO) mice and wild type (WT) ones. The isometric force induced by various concentrations of high KCl was lower in KO than in WT both in aorta and in vas deferens. The length-force relations were compared between KO and WT. The active isometric force in KO was significantly lower at most muscle lengths examined than in WT without the change in resting force both in aorta and in vas deferens. In vas deferens, the rate of force development after quick release in length at the peak force was significantly faster in KO than in WT. The above results show that the force development is lower and the rate of cross-bridge cycle is faster in KO mice than in WT ones, suggesting that calponin plays basic roles in the control of the contraction of smooth muscle.

Neuroblastoma is a tumor that is derived from the neural crest. Recent studies demonstrated that several human neuroblastoma cell lines exhibit at least three morphologic types: neuroblastic (N)-type, substrate-adhesive (S)-type and intermediate (I)-type cells. However, the origin of the S-type cells has not been clearly identified. In this study, the expressions of smooth muscle-specific proteins (desmin, alpha-smooth muscle actin, basic calponin and the smooth muscle myosin heavy-chain isoforms of SM1 and SM2) in three parent and four cloned neuroblastoma cell lines, composed of S-type cells, were examined by indirect immunofluorescence, Western blot and/or by reverse transcription-polymerase chain reaction (RT-PCR). Desmin was found in two of the seven cell lines, and alpha-smooth muscle actin and basic calponin were detected in all of seven of the cell lines. In three parent cell lines and one cloned cell line composed of N-type cells, none of three smooth muscle-specific proteins were detected. In smooth muscle myosin heavy-chain isoforms, SM1 was detected in two parent cell lines composed of S-type cells (MP-N-MS and KP-N-YS) by immunofluorescence, Western blot and/or by RT-PCR, whereas the SM2 isoform was detected in one parent cell line (MP-N-MS) by RT-PCR. These findings indicate that S-type cells have either the immature or mature smooth muscle cell phenotype, and neural crest cells very likely have the ability of to differentiate into smooth muscle cells in the human system.

1 The role of h1-calponin in regulating the contractile properties of smooth muscle was investigated in bladder and vas deferens of mice carrying a targeted mutation in both alleles designed to inactivate the basic calponin gene. These calponin knockout (KO) mice displayed no detectable h1-calponin in their smooth muscles. 2 The amplitudes of Ca2+ sensitization, force and Ca2+ sensitivity were not significantly different in permeabilized smooth muscle of KO compared with wild-type (WT) mice, nor were the delays in onset and half-times of Ca2+ sensitization, initiated by flash photolysis of caged GTPγS, different. 3 The unloaded shortening velocity (Vus) of thiophosphorylated fibres was significantly (P < 0.05) faster in the smooth muscle of KO than WT animals, but could be slowed by exogenous calponin to approximate WT levels; the concentration dependence of exogenous calponin slowing of Vus was proportional to its actomyosin binding in situ. 4 Actin expression was reduced by 25-50%, relative to that of myosin heavy chain, in smooth muscle of KO mice, without any change in the relative distribution of the actin isoforms. 5 We conclude that the faster Vus of smooth muscle of the KO mouse is consistent with, but does not prove without further study, physiological regulation of the crossbridge cycle by calponin. Our results show no detectable role of calponin in the signal transduction of the Ca2+-sensitization pathways in smooth muscle.

To elucidate the function of calponin in intact contracting smooth muscle cells in vivo, we generated mice with a mutated basic calponin (h1) locus (Yoshikawa et al., Genes Cells 3, 685–695, 1998). Crossbridge cycling rates were estimated in aortic smooth muscle by the force redevelopment following an isometric step shortening as a function of time after K+ depolarization. Evidence is presented that calponin is involved in the inhibition of shortening velocity in the tonic phase of contraction. The phosphorylation levels of myosin regulatory light chain and cytosolic calcium concentrations were not significantly different in paired comparisons between calponin-deficient (−/−) and wild-type (+/+) muscles at any time point after stimulation. The force-velocity relationships in vas deferens smooth muscle showed that the maximum shortening velocity of −/− muscle was significantly faster than that of +/+ muscle. There was no change in the length-force relationships in both −/− and +/+ muscles of aorta and vas deferens. The results suggest that calponin plays a role in regulation of the crossbridge cycling and that it may be responsible for reduced shortening velocity during a maintained contraction of mammalian smooth muscle.

Malignant rhabdoid tumor (MRT) is a rare and extremely aggressive malignant tumor in childhood. In this study, an MRT cell line, designated KP-MRT-NS, was established from the ascitic fluid taken from an 11-month-old girl, whose tumor had originated from the left kidney. Ultrastructural findings demonstrated the typical aggregation of whorls of intermediate filaments. Chromosome constitution was described as 46, XX, add (10)(q26)[17]/46, idem, dis (1;2)(q22;q31)[3] based on ISCN (1995) and a del (22)(q11.2) was not found in this cell line. The origin of MRT is controversial, various cellular origins having been proposed because of the phenotypic diversity of MRT. Therefore, in this study, to clarify the origin of MRT, the expressions of cytoplasmic proteins including smooth-muscle-specific proteins (alpha-smooth-muscle actin, basic calponin, smooth-muscle-myosin-heavy-chain isoforms of SM1 and SM2) in the primary-MRT tissue and cell line were analyzed. In the primary-tumor tissue, the expressions of neurofilament, vimentin and alpha-smooth-muscle actin were demonstrated by indirect immunofluorescence. In the KP-MRT-NS cell line, the expression of neurofilament, alpha-smooth-muscle actin, basic calponin and smooth-muscle-myosin heavy chain of SM1 and SM2 isoforms was revealed by immunofluorescence, Western blot and/or reverse transcriptase-polymerase chain reaction (RT-PCR). MyoD1 mRNA, determined as a skeletal-muscle-cell lineage marker, was not expressed in the primary-tumor tissue or in the KP-MRT-NS cell line. According to our findings, the MRT cells are of both neural and smooth-muscle cell phenotypes, and support the neural-crest origin of MRT.

Subarachnoid hemorrhage (SAH) often leads to a long-term narrowing of cerebral artery called vasospasm. To understand the molecular mechanisms in vasospasm, signal transduction of tyrosine kinase pathway and phosphorylation of myosin light chain (MLC) and calponin (CaP) in the basilar artery were studied. Vasospasm was produced in the canine basilar artery by a two-hemorrhage method, and vasocontraction was induced by a local application of KCl or serotonin to the basilar artery after a transclival exposure. Intracellular substrates of tyrosine kinase pathway, including Shc, Raf1, and extracellular-regulated kinases in the basilar artery, were activated after SAH, and the activation of Shc suggests stimulation of signal transductions from tyrosine kinase receptors, G-coupled receptors, or both. The activation of tyrosine kinase pathway in vasospasm also was supported by dose-dependent dilation of the spastic basilar artery on days 0 and 7 by topical application of genistein, a tyrosine kinase inhibitor, and associated marked inhibition of tyrosine phosphorylation of intracellular substrates, including Shc. In addition, the generation of protein kinase M, catalytic fragment of protein kinase Cα (PKCα), in vasospasm on days 0 and 7 was inhibited in response to genistein, indicating an inactivation of μ-calpain. It is suggested, therefore, that the reversal of vasospasm by genistein is closely associated with the restoration of intracellular Ca2+ levels. However, the increased activities of Raf1 and extracellular-regulated kinases in vasospasm were declined on day 7 compared with those on day 0 or 2, suggesting that the activation of tyrosine kinase pathway is more closely associated with the early stage of vasospasm than with the late stage of vasospasm. The analysis by pyrophosphate polyacrylamide gel electrophoresis (PPi-PAGE) demonstrated three MLC bands in vasospasm on days 2 and 7, as well as in KCl- and serotonin-induced vasocontraction. Since PPi-PAGE resolves smooth muscle MLC into three bands in the MLC kinase (MLCK)-mediated phosphorylation and into a single band in the PKC-mediated phosphorylation based on the phosphorylation state, the current results suggest that MLC in vasospasm is phosphorylated by MLCK but not by PKC. In basilar artery, CaP was significantly down-regulated, and in addition, significantly phosphorylated on serine and threonine residues only in vasospasm on days 2 and 7. Although the significance of CaP phosphorylations in vivo still is controversial, CaP down-regulation and phosphorylation may attenuate the inhibition of Mg2+-ATPase activity by CaP and induce a potential enhancement of smooth muscle contractility in delayed vasospasm. Since CaP is phosphorylated vivo by PKC, activated PKC in vasospasm may phosphorylate CaP. Thus, SAH stimulates tyrosine kinase pathway to increase intracellular Ca2+ and activate PKC, and the former activates MLCK to phosphorylate MLC, whereas the latter phosphorylates CaP but not MLC.

Purpose.Histogenesis of synovial sarcoma remains controversial and reliable molecular markers for diagnosis are necessary. Expression of basic calponin, a smooth muscle differentiation-specific actin-binding protein, was studied in synovial sarcoma.Subjects and Methods.The basic calponin gene and the gene product were analyzed by reverse transcription PCR analysis (RT-PCR) and immunohistochemistry in 14 synovial sarcomas and a human synovial sarcoma cell line (HS-SY-II).Results and Discussion.Immunoreactivity for basic calponin was detected in the cytoplasm of 6 synovial sarcomas (43% positive). In the basic calponin-positive tumors and the HS-SY-II cells, expression for smooth muscle-specific genes, including basic calponin and SM22α , was detected by RT-PCR, suggesting a lineage relationship between synovial sarcoma cells and smooth muscle-like mesenchymal cells.Conclusions.A subset of synovial sarcomas expressing the basic calponin gene and the gene product were identified. The basic calponin may have potential utility as a novel molecular marker identifying certain synovial sarcomas.

Background Calponin is a calmodulin-and actin-binding protein expressed in smooth muscle. It promotes actin polymerization and inhibits actin-activated myosin ATPase activity. Despite the molecular and functional characterization of calponin in vitro, the physiological role of calponin in vivo has not been clarified. Results We investigated the in vivo function of smooth muscle calponin (also called basic calponin or calponin h1) by generating mice carrying a targeted mutation in both alleles of the calponin gene. Mice lacking basic calponin expression displayed enhanced ectopic bone formation in vivo, induced by recombinant human bone morphogenetic protein-2 (rhBMP-2), and an augmentation of the degree of osteoblastic differentiation of embryonic mesenchymal cells when they were stimulated by rhBMP-2. Basic calponin messenger RNA was shown to be expressed in developing and healing bone tissues, and in undifferentiated MC3T3-E1 osteoblasts. An examination of the skeletons of mutated mice showed an early onset of cartilage formation and ossification, and increased postnatal bone formation characterized by an increase in the number of activated periosteal osteoblasts. Bone fracture healing was accelerated in mutated mice. Conclusion This is the first demonstration of animals with enhanced BMP responsiveness in host cells, suggesting that endogenous basic calponin may play a negative role in an osteogenic programme.

The basic calponin gene is a smooth muscle differentiation-specific gene that encodes an actin-binding protein involved in the regulation of smooth muscle contractility. We studied the expression of the calponin gene in 8 human osteosarcoma cell lines and 17 primary human osteosarcoma tissues by RT-PCR analysis. We also analyzed mRNA expression of smooth muscle-specific genes including SM22alpha, caldesmon and alpha-actin, and for neutral and acidic calponin isoforms. The genes were expressed at various levels by osteosarcoma cell lines and tissues of diverse histological subtypes. The basic calponin protein of an expected size was detected in osteosarcoma cell lines by immunoblot analysis and was localized by immunohistochemistry in the cytoplasm of the tumor cells in osteosarcoma tissues. Survival was found to be significantly increased in patients whose tumors exhibited basic calponin expression, compared with those with no expression. Alterations in the expression of other markers examined were not correlated with prognosis. Our results suggest that the basic calponin gene product may be a novel prognostic variable in patients with osteosarcoma.

Calponin (h1 isoform) was characterized as a smooth muscle specific, actin-, tropomyosin-, calmodulin-binding protein and described as a factor which inhibits contraction. H2-calponin, encoded by a different gene from h1-calponin, was identified from the smooth muscles of mouse and pig. However, non-muscle calponin analogues have recently been reported in rat and pig brains. Here we show the presence of calponin expressed in human skin tissue and in cultured human keratinocytes using polyclonal antibodies to bovine aortic smooth muscle calponin. Western blot analysis demonstrated that calponin with a molecular weight around 36000 existed in extracts of keratinocytes. Immunofluorescence microscopy displayed the localization of calponin in the cytoplasm of the basal cells in situ, and along the cell-to-cell borders in cultured human keratinocytes maintained in standard calcium medium. Furthermore, according to RT-PCR analysis using human h1- and h2-calponin-specific primers, calponin expressed by cultured human keratinocytes was identified as the h2 isoform. We demonstrated the presence of h2-calponin in human keratinocytes, and it might play a role in the structural organization of actin cytoskeleton at the cytoplasmic region of cell-to-cell junctions of keratinocytes.

The safety and anti-tumor effect of oncolytic virus have been reported in a clinical study conducted in Japan. We have engineered a novel multimutated tumor-specific oncolytic herpes virus, harboring a smooth muscle-specific calponin promoter. Since tumor cells present in a hypoxic environment are known to be resistant to chemotherapy and radiotherapy, we also engineered a novel oncolytic herpes virus targeting a specific tumor microenvironment, which harbors a gene encoding a fusion protein of oxygen-dependent degradation (ODD) domain of HIF1α and ICP4, a master viral transcription factor required for replication. The recombinant virus selectively replicates in and disrupts the target tumor cells, including human sarcoma and malignant mesothelioma cells which are unresponsive to chemotherapy and molecular targeted therapy. We confirmed significant anti-tumor effects of the novel viruses in vivo in an allogeneic experimental model of an undifferentiated pleomorphic sarcoma (malignant fibrous histiocytoma; MFH) spontaneously generated in immunocompetent Fischer rats. Our viruses, manufactured in the Master Virus Seed Stock in the Good Manufacturing Practice facility will become novel agents that enable tumor cells unresponsive to conventional treatment to be disrupted.

cDNA clones encoding a calponin isoform with 309 amino acids have been isolated from human heart. The deduced amino acid polypeptide (M(r) 33,697) showed a neutral isoelectric point of 7.1. The mRNA, expressed in cultured smooth muscle cells as well as in fibroblasts, vascular endothelial cells, and keratinocytes, contains a 3' untranslated region of 1.2 kilobases that includes an Alu repetitive sequence in the antisense direction. On the basis of the nucleotide sequence identity to an expressed sequence tag, HUM21ES93 [Cheng, J.-F., Boyartchuk, V., and Zhu, Y. (1994) Genomics 23, 75-84], the human neutral calponin gene is assigned to chromosome 21q11.1. The amino acid sequence indicates that this protein is the human equivalent of mouse calponin-h2 (94.8% identity) [Strasser, P., Gimona, M., Moessler, H., Herzog, M., and Small, J.V. (1993) FEBS Lett. 330, 13-18]. Three tandem repeats of 29 amino acids, a Vav-homologous region and an actin-binding sequence, originally identified in the basic calponin isoform, are conserved. There are two consensus phosphorylation sites for tyrosine kinase. An immunoreactive form of the neutral calponin appears to be localized with vinculin in the cell-to-cell junctions of cardiomyocytes. Mouse calponin-h2 is also expressed in both embryonic and adult heart. These results indicate that the human neutral calponin is a non-smooth muscle isoform, and may play a physiological role in cytoskeletal organization.

The human smooth muscle cell (SMC) calponin gene, which is composed of seven exons and six introns, spanning an approximately 11.2 kilobase (kb) genomic DNA, has been isolated and characterized by sequence analysis. As determined by primer extension mapping and rapid amplification of mRNA transcripts, a major transcription start site of the calponin gene is located at 101 base-pairs (bp) upstream of the ATG start codon. A striking feature of the 5'-flanking, 5'-untranslated, and amino-terminal protein coding regions is the presence of an Alu repetitive sequence and a stretch of DNA sequence identified using a methyl CpG-binding protein affinity column [Cross, S.H., Charlton, J.A., Nan, X., and Bird, A.P. (1994) Nature Genet, 6,236-244]. The results of this study provide a new insight into the molecular mechanism underlying regulation of SMC-lineage specific gene expression.

Calponin is a thin filament-associated protein that is implicated in the regulation and maintenance of smooth muscle contraction. Molecular cloning of chicken gizzard calponin indicated the presence of two isoforms, alpha and beta, the expression of the alpha-isoform being uniformly more abundant in various smooth muscle tissues [Takahashi, K. & Nadal-Ginard, B. (1991) J. Biol. Chem. 266, 13284-13288]. For the long-range goal of understanding of the structure and function of calponin, we have started bacterial expression and site-directed mutagenesis of alpha calponin. The amino acid composition and N-terminal sequence of the recombinant alpha calponin were found to be identical to those deduced from its nucleotide sequence. Recombinant alpha calponin is capable of binding to calmodulin, troponin C, tropomyosin, and actin, and of inhibiting skeletal muscle acto-subfragment-1 ATPase activity. A mutant alpha calponin with a replacement in the putative inhibitory region (residues 146-171) has impaired ability to inhibit the acto-subfragment-1 ATPase activity, suggesting that this region of calponin may be involved in the modulation of the actin-myosin interactions.

In this paper, some results of field observation concerning the anual and short-term behavior of salt intrusion in Lake Abasiri is reported. From the data analysis, we found that this lake has two layers of salt and fresh water vertically and that the lower layer of salt water is gradually ascending in this decade and it has no soluted oxygen. In the field observation in the river, we found that the salt water intruded into the lake in the form of salt wedge. Because of the occurence of record breaking heavy rainfall in this catchement area just before the observation, the thickness of the upper layer became from about 5 to 7 meter.In order to make clear the mechanism of long-term trends of the lake, we proposed the model of tank-model type. In the final section of this paper, we compared the numerical results with the field data and we found the good agreement.

Calponin inhibited the actin-activated myosin MgATPase activity in a dose-dependent manner without affecting the phosphorylation level of myosin light chain. This inhibition was Ca2(+)-independent. The decrease in enzymatic activity of myosin was correlated with binding of calponin to actin-tropomyosin filaments. Caldesmon showed a further inhibition of the calponin-induced inhibition of MgATPase activity of the thiophosphorylated myosin. Calponin-induced inhibition of the myosin MgATPase activity was reversed by the addition of calmodulin only in the presence of Ca2+. These results suggest that calponin acts as an inhibitory component of smooth muscle thin filaments.

The gene and cDNA clones encoding smooth muscle calponin (also called basic calponin or calponin h1) have been isolated and characterized. The NH2-terminal region of calponin shares homology with a putative negative regulatory domain of the GDP-GTP exchanger for Rho-like GTP-binding proteins. Expression of the basic isoform of calponin is downmodulated in proliferating smooth muscle cells of atherosclerotic neointima and injured arterial media. Transfer of the recombinant human calponin gene into rabbit balloon-injured arteries or rat transgenic arteries in vivo increased calponin expression by smooth muscle cells and suppressed arterial myointimal hyperplasia. The cellular distribution of calponin has been shown to be dramatically changed upon stimulation. with an agonist known to activate protein kinase C in vascular smooth muscle cells. In addition to regulation of the contraction-relaxation cycle of smooth muscle, the findings reviewed here suggest that calponin might also be involved in the control of adhesion, migration, and proliferation of smooth muscle cells.