Mismatch repair systems might facilitate the chromosomal recombination induced by N-nitrosodimethylamine, but not by N-nitrosodiethylamine, in Drosophila.

Mismatch repair (MMR) systems play important roles in maintaining the high fidelity of genomic DNA. It is well documented that a lack of MMR increases the mutation rate, including base exchanges and small insertion/deletion loops; however, it is unknown whether MMR deficiency affects the frequency of chromosomal recombination in somatic cells. To investigate the effects of MMR on chromosomal recombination, we used the Drosophila wing-spot test, which efficiently detects chromosomal recombination. We prepared MMR (MutS)-deficient flies (spel1(-/-)) using a fly line generated in this study. The spontaneous mutation rate as measured by the wing-spot test was slightly higher in MutS-deficient flies than in wild-type (spel1(+/-)) flies. Previously, we showed that N-nitrosodimethylamine (NDMA)-induced chromosomal recombination more frequently than N-nitrosodiethylamine (NDEA) in Drosophila. When the wing-spot test was performed using MMR-deficient flies, unexpectedly, the rate of NDMA-induced mutation was significantly lower in spel1(-/-) flies than in spel1(+/-) flies. In contrast, the rate of mutation induced by NDEA was higher in spel1(-/-) flies than in spel1(+/-) flies. These results suggest that in Drosophila, the MutS homologue protein recognises methylated DNA lesions more efficiently than ethylated ones, and that MMR might facilitate mutational chromosomal recombination due to DNA double-strand breaks via the futile cycle induced by MutS recognition of methylated lesions.

Ryanodine receptor mutations (G4946E and I4790K) differentially responsible for diamide insecticide resistance in diamondback moth, Plutella xylostella L.

This study examined diamondback moth (Plutella xylostella) strains showing high-level resistance to cyantraniliprole (KA17 strain) and to flubendiamide and chlorantraniliprole (KU13 strain). The LC50 value of the KA17 strain against cyantraniliprole was ca. 100-fold higher than that of the KU13 strain. The KA17 strain also exhibited high-level resistance to chlorantraniliprole and flubendiamide equivalent to those in the KU13 strain. The KU13 strain showed a higher LC50 value against cyantraniliprole than the susceptible strains. However, the LC50 value of the KU13 strain against cyantraniliprole was below the agriculturally recommended concentration. Subsequent QTL analysis using ddRAD-seq identified the resistance responsible regions of the KA17 and KU13 strains with different diamide resistance profiles. Ryanodine receptor (RyR) gene was included in the identified regions. Single nucleotide polymorphism calling in the RyR gene using RNA-seq found previously reported G4946E (amino acid mutation from glycine to glutamic acid at amino acid position 4946) and novel I4790K (amino acid mutation from isoleucine to lysine at amino acid position 4790) mutations, respectively, in the RyR of the KU13 and KA17 strains. Functional significance of I4790K in the resistance was confirmed in calcium imaging of the human embryonic kidney 293T cell line expressing Bombyx mori RyR with the mutation. This reporting is the first describing I4790K as a fundamental mechanism responsible for the resistance to the diamides including cyantraniliprole. From this study, we also report up-regulated expression of some degradation enzymes and that of the RyR gene in the KA17 and KU13 strains based on results of RNA-seq data analysis.

DNA Origami Scaffolds as Templates for Functional Tetrameric Kir3 K+ Channels.

In native systems, scaffolding proteins play important roles in assembling proteins into complexes to transduce signals. This concept is yet to be applied to the assembly of functional transmembrane protein complexes in artificial systems. To address this issue, DNA origami has the potential to serve as scaffolds that arrange proteins at specific positions in complexes. Herein, we report that Kir3 K+ channel proteins are assembled through zinc-finger protein (ZFP)-adaptors at specific locations on DNA origami scaffolds. Specific binding of the ZFP-fused Kir3 channels and ZFP-based adaptors on DNA origami were confirmed by atomic force microscopy and gel electrophoresis. Furthermore, the DNA origami with ZFP binding sites nearly tripled the K+ channel current activity elicited by heterotetrameric Kir3 channels in HEK293T cells. Thus, our method provides a useful template to control the oligomerization states of membrane protein complexes in vitro and in living cells.

New criteria to predict tumor recurrence in invasive ductal carcinoma of the breast.

Incidence of triple-negative breast cancer (TNBC), which is cancer without expression of ER, PgR, and HER2, and nuclear grade (NG) are closely correlated with malignant potential of breast cancer. However, criteria to determine aggressiveness of breast cancer based on these factors have not been elucidated. The aim of this study was to create criteria using these factors to predict tumor recurrence in invasive ductal carcinoma (IDC) of the breast. One hundred and seventy-nine patients with IDC of the breast, which had been treated by surgical resection, were included. One point was added for each factor of the two categories of TNBC and NG 3. The sum of the scores (TGS 0, 1, or 2) was calculated. Significant difference was observed between TGS and the incidence of tumor recurrence (P < 0.0001). Moreover, significant differences were observed regarding relapse-free survival (RFS) between patients with TGS 0 and TGS 1 (P < 0.0001) and patients with TGS 1 and TGS 2 (P = 0.024). TGS might contain a clinical advantage as a useful predictor for tumor recurrence of IDC of the breast and could classify prognosis of the patients with a preferable stratification.

Pedunculated hamartomatous polyp of the appendix: report of a case.

We experienced a rare case of a pedunculated polyp of the appendix, which had been incidentally found by preventive appendectomy performed when providing surgical treatment for rectal carcinoma. A pathological investigation of this polypoid lesion demonstrated branches of fibro-muscular stalks connecting with the lamina muscularis covered by a hyperplastic mucosa, which proved to be consistent with the features of hamartoma. The patient had no external characteristics of Peutz-Jeghers syndrome, including mucocutaneous pigmentation and gastrointestinal polyposis, observed by endoscopy. This case is considered to be a Peutz-Jeghers type polyp of the appendix with a pedunculated form, which is very rare.

Unilateral multicentric cancer of the breast containing two different histopathological types: Case report.

Unilateral multicentric cancer of the breast containing a tumor with a specific histopathological type is comparatively rare. We, herein, report a case of an unilateral multicentric cancer of the breast containing two separate tumors diagnosed as a papillo-tubular carcinoma and an invasive lobular carcinoma. In addition to the difference in histological type, these two tumors also had different patterns of expression of hormonal receptors reflecting the cellular aggressiveness. Therfore, these tumors may be formed through multicentric tumorigenesis.

Analysis of the impact of the body mass index in patients with gastric carcinoma.

BACKGROUND: Body mass index (BMI) has been suggested to provide clinicopathological information in tumor development and progression in patients with gastric carcinoma. METHODS: The correlation of BMI with clinicopathological features and operation-related factors was analyzed in 308 patients with gastric carcinoma who had undergone distal or total gastrectomy. RESULTS: There was no significant correlation of obesity, indicated by a high value of BMI, with tumor-related factors including survival, or with operation-related factors. On the other hand, more advanced tumors and worse preoperative nutritional and immunological conditions were found in patients with a lower value of BMI. CONCLUSIONS: BMI might be a representation of the physical condition brought about by the extent of tumor progression rather than a factor influencing the factors related to gastric carcinoma.

[High incidence-rate of oral mucositis in breast cancer patients receiving anthracycline-based chemotherapy (FEC100)].

BACKGROUND: Oral mucositis is a frequent complication, but is poorly studied among patients with solid tumors. The purpose of this study is to clarify the incidence rate of oral mucositis in Japanese breast cancer patients receiving anthracycline-based chemotherapy(FEC100). METHODS: From June 2007 to July 2008, 61 breast cancer patients eligible for this study received anthracycline-based chemotherapy(FEC100: 5-FU 500mg/m / / 2, epirubicin 100 mg/m2, cyclophosphamide 500 mg/m2)at National Kyushu Cancer Center and Iwate Medical University Hospital. The incidence rate and grade of oral mucositis were evaluated in these patients. RESULTS: The cumulative incidence of oral mucositis was about 50%. Episodes of oral mucositis were more common during courses with febrile neutropenia than during courses without it(75. 0% vs 44. 9%, p=0. 12). The reduction of oral mucositis was only 13. 6% after administering the steroidal ointment and/or mouthwash, including sodium azulene sulfonate. CONCLUSIONS: New methods are needed to prevent and treat oral mucositis in patients receiving anthracycline- based chemotherapy(FEC100).

Pathological Prognostic Score: a rational criteria to stratify survival in colorectal carcinoma.

PURPOSE: The aim of this study is to certify a rationality of Pathological Prognostic Score (PPS) in determining the prognosis of patients with colorectal carcinoma. METHODS: Three hundred and thirty-one patients with colorectal carcinoma, which had been treated by surgical resection, were enrolled. One point was added for each element among four tumor-related pathological factors of depth of tumor, lymph node metastasis, venous invasion, and lymphatic invasion. PPS was determined by an aggregate of the points. RESULTS: There existed a significant difference both between survivals of patients with PPS 0 or 1 and 2 or 3 (P = 0.0005) and between survivals of patients with PPS 2 or 3 and 4 (P < 0.0001). CONCLUSIONS: PPS could be an easy and useful criteria to stratify prognosis of patients with colorectal carcinoma.

Squamous cell carcinoma of the breast.

Squamous cell carcinoma (SCC) of the breast is comparatively rare. Because of the small number of investigations based on sufficient population size, its clinicopathological features have not been fully elucidated. We report herein a patient with SCC of the breast who underwent surgical resection and whose therapeutic options are now considered.

Serum appearance of anti-p53 antibody in triple negative breast cancer.

BACKGROUND: Significance of serum appearance of anti-p53 antibody in breast cancer has not been discussed fully. METHODS: The relationship between serum appearance of anti-p53 antibody and the clinicopathological features was investigated on the basis of the data of 42 women with breast cancer who had been treated with surgical resection. RESULTS: The proportion of tumors with histological grade 3 was significantly higher among tumors with serum appearance of anti-p53 antibody (P = 0.020). The proportion of triple negative tumors was also significantly higher among tumors with serum appearance of anti-p53 antibody (P = 0.003). CONCLUSIONS: Preoperative appearance of anti-p53 antibody in sera can be correlated with the incidence of triple negative breast cancer and could therefore help identify tumors with aggressive potential.

The prognostic nutritional index can be a prognostic indicator in colorectal carcinoma.

PURPOSE: Preoperative assessments regarding a patient's immunological and nutritional condition are required to predict the outcomes of patients with malignant tumors. The aim of the current study was to clarify the significance of Onodera's prognostic nutritional index (OPNI), which can simply account for the immunological and nutritional conditions, in patients with colorectal carcinoma. METHODS: The correlations of the preoperative OPNI value with clinicopathological features were examined in 219 patients with colorectal carcinoma who had been surgically treated. RESULTS: Not only the tumor stage (P = 0.028) and venous invasion (P = 0.002), but also an OPNI of less than 40 (P = 0.002) were found to be independently correlated with a worse prognosis of patients with colorectal carcinoma. CONCLUSION: The preoperative OPNI can be used as a simple prognostic indicator in colorectal carcinoma.

Primary neuroendocrine carcinoma of the breast: report of a case.

Primary neuroendocrine carcinoma (NEC) of the breast appears to be a rare neoplasm. Due to the limited number of the cases, a definitive therapeutic option for the disease has not yet been established. We herein report the case of a 57-year-old female patient with primary NEC of the breast who underwent a surgical resection and for whom the suitable adjuvant therapy is now being considered.

[Prominent efficacy from capecitabine mono-therapy for a case with lymphangiosis carcinomatosa derived from recurrent breast cancer].

We met a 61-year-old woman who had been suffering from dyspnea due to pleuritis carcinomatosa derived from recurrent breast cancer for which no former chemotherapy had been effective. Capecitabine mono-therapy could exert a drastic anticancer effect for lymphangiosis carcinomatosa causing more severe symptoms of dyspnea. This therapeutic option could be selected for patients with recurrent breast cancer for which the first- or second-line chemotherapy could not provide a sufficient anti-cancer efficacy and possibly serve to sustain the life quality of the patients.

Immunohistochemical expression of epidermal growth factor receptor in breast cancer.

BACKGROUND: Molecular-targeting drugs able to treat breast cancer expressing epidermal growth factor receptor (EGFR) would be clinically valuable. The aim of the current study was to determine the further significance of immunohistochemical expression of EGFR in breast cancer. METHODS: The immunohistochemical expression of EGFR was examined in 37 women with breast cancer who had been treated with surgical resection. Relationship of EGFR expression and clinicopathological characteristics was investigated. RESULTS: EGFR expression proved to be comparatively more frequent among triple-negative (estrogen receptor negative, progesterone receptor negative, and human epidermal growth factor receptor 2 negative) breast cancers (P = 0.067). CONCLUSIONS: Knowledge of a patient's immunohistochemical EGFR expression, which could be one of the key molecular findings related to molecular-targeting therapy, might be useful information to treat triple-negative breast cancer.

Rab3-interacting molecule gamma isoforms lacking the Rab3-binding domain induce long lasting currents but block neurotransmitter vesicle anchoring in voltage-dependent P/Q-type Ca2+ channels.

Assembly of voltage-dependent Ca(2+) channels (VDCCs) with their associated proteins regulates the coupling of VDCCs with upstream and downstream cellular events. Among the four isoforms of the Rab3-interacting molecule (RIM1 to -4), we have previously reported that VDCC beta-subunits physically interact with the long alpha isoform of the presynaptic active zone scaffolding protein RIM1 (RIM1alpha) via its C terminus containing the C(2)B domain. This interaction cooperates with RIM1alpha-Rab3 interaction to support neurotransmitter exocytosis by anchoring vesicles in the vicinity of VDCCs and by maintaining depolarization-triggered Ca(2+) influx as a result of marked inhibition of voltage-dependent inactivation of VDCCs. However, physiological functions have not yet been elucidated for RIM3 and RIM4, which exist only as short gamma isoforms (gamma-RIMs), carrying the C-terminal C(2)B domain common to RIMs but not the Rab3-binding region and other structural motifs present in the alpha-RIMs, including RIM1alpha. Here, we demonstrate that gamma-RIMs also exert prominent suppression of VDCC inactivation via direct binding to beta-subunits. In the pheochromocytoma PC12 cells, this common functional feature allows native RIMs to enhance acetylcholine secretion, whereas gamma-RIMs are uniquely different from alpha-RIMs in blocking localization of neurotransmitter-containing vesicles near the plasma membrane. Gamma-RIMs as well as alpha-RIMs show wide distribution in central neurons, but knockdown of gamma-RIMs attenuated glutamate release to a lesser extent than that of alpha-RIMs in cultured cerebellar neurons. The results suggest that sustained Ca(2+) influx through suppression of VDCC inactivation by RIMs is a ubiquitous property of neurons, whereas the extent of vesicle anchoring to VDCCs at the plasma membrane may depend on the competition of alpha-RIMs with gamma-RIMs for VDCC beta-subunits.

Ca2+ influx and protein scaffolding via TRPC3 sustain PKCbeta and ERK activation in B cells.

Ca(2+) signaling mediated by phospholipase C that produces inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] and diacylglycerol (DAG) controls lymphocyte activation. In contrast to store-operated Ca(2+) entry activated by Ins(1,4,5)P(3)-induced Ca(2+) release from endoplasmic reticulum, the importance of DAG-activated Ca(2+) entry remains elusive. Here, we describe the physiological role of DAG-activated Ca(2+) entry channels in B-cell receptor (BCR) signaling. In avian DT40 B cells, deficiency of transient receptor potential TRPC3 at the plasma membrane (PM) impaired DAG-activated cation currents and, upon BCR stimulation, the sustained translocation to the PM of protein kinase Cbeta (PKCbeta) that activated extracellular signal-regulated kinase (ERK). Notably, TRPC3 showed direct association with PKCbeta that maintained localization of PKCbeta at the PM. Thus, TRPC3 functions as both a Ca(2+)-permeable channel and a protein scaffold at the PM for downstream PKCbeta activation in B cells.

A pathogenic C terminus-truncated polycystin-2 mutant enhances receptor-activated Ca2+ entry via association with TRPC3 and TRPC7.

Mutations in PKD2 gene result in autosomal dominant polycystic kidney disease (ADPKD). PKD2 encodes polycystin-2 (TRPP2), which is a homologue of transient receptor potential (TRP) cation channel proteins. Here we identify a novel PKD2 mutation that generates a C-terminal tail-truncated TRPP2 mutant 697fsX with a frameshift resulting in an aberrant 17-amino acid addition after glutamic acid residue 697 from a family showing mild ADPKD symptoms. When recombinantly expressed in HEK293 cells, wild-type (WT) TRPP2 localized at the endoplasmic reticulum (ER) membrane significantly enhanced Ca(2+) release from the ER upon muscarinic acetylcholine receptor (mAChR) stimulation. In contrast, 697fsX, which showed a predominant plasma membrane localization characteristic of TRPP2 mutants with C terminus deletion, prominently increased mAChR-activated Ca(2+) influx in cells expressing TRPC3 or TRPC7. Coimmunoprecipitation, pulldown assay, and cross-linking experiments revealed a physical association between 697fsX and TRPC3 or TRPC7. 697fsX but not WT TRPP2 elicited a depolarizing shift of reversal potentials and an enhancement of single-channel conductance indicative of altered ion-permeating pore properties of mAChR-activated currents. Importantly, in kidney epithelial LLC-PK1 cells the recombinant 679fsX construct was codistributed with native TRPC3 proteins at the apical membrane area, but the WT construct was distributed in the basolateral membrane and adjacent intracellular areas. Our results suggest that heteromeric cation channels comprised of the TRPP2 mutant and the TRPC3 or TRPC7 protein induce enhanced receptor-activated Ca(2+) influx that may lead to dysregulated cell growth in ADPKD.

Molecular characterization of flubendiamide sensitivity in the lepidopterous ryanodine receptor Ca(2+) release channel.

Flubendiamide is a benzenedicarboxamide derivative that shows selective insecticidal activity against lepidopterous insects. The specific modulatory effects of flubendiamide on ryanodine binding in insect muscle microsomal membranes suggest that the ryanodine receptor (RyR) Ca(2+) release channel is a primary target of flubendiamide. However, the molecular mechanisms underlying the species-specific action of flubendiamide are unclear. We have cloned cDNA encoding a novel RyR from the lepidopterous silkworm RyR (sRyR) and tested the sensitivity to flubendiamide of the recombinant sRyR in HEK293 cells. Confocal localization studies and Ca(2+) imaging techniques revealed that sRyRs form Ca(2+) release channels in the endoplasmic reticulum. Importantly, flubendiamide induced release of Ca(2+) through the sRyR, but not through the rabbit RyR isoforms. Photoaffinity labeling of sRyR deletion mutants using a photoreactive derivative revealed that flubendiamide is mainly incorporated into the transmembrane domain (amino acids 4111-5084) of the sRyR. The rabbit cardiac muscle isoform RyR2 (rRyR2) and the RyR mutant carrying a replacement of the transmembrane domain (residues 4084-5084) with its counterpart sequence from rRyR2 (residues 3936-4968) were not labeled by the photoreactive compound. This replacement in the sRyR significantly impaired the responses to flubendiamide but only marginally reduced the sensitivity to caffeine, a general RyR activator. Furthermore, deletion of the N-terminal sequence (residues 183-290) abolished the responses of the sRyR to flubendiamide but not the sensitivity to caffeine. Our results suggest that the transmembrane domain plays an important role in the formation of an action site for flubendiamide, while the N-terminus is a structural requirement for flubendiamide-induced activation of the sRyR.

Selective and direct inhibition of TRPC3 channels underlies biological activities of a pyrazole compound.

Canonical transient receptor potential (TRPC) channels control influxes of Ca(2+) and other cations that induce diverse cellular processes upon stimulation of plasma membrane receptors coupled to phospholipase C (PLC). Invention of subtype-specific inhibitors for TRPCs is crucial for distinction of respective TRPC channels that play particular physiological roles in native systems. Here, we identify a pyrazole compound (Pyr3), which selectively inhibits TRPC3 channels. Structure-function relationship studies of pyrazole compounds showed that the trichloroacrylic amide group is important for the TRPC3 selectivity of Pyr3. Electrophysiological and photoaffinity labeling experiments reveal a direct action of Pyr3 on the TRPC3 protein. In DT40 B lymphocytes, Pyr3 potently eliminated the Ca(2+) influx-dependent PLC translocation to the plasma membrane and late oscillatory phase of B cell receptor-induced Ca(2+) response. Moreover, Pyr3 attenuated activation of nuclear factor of activated T cells, a Ca(2+)-dependent transcription factor, and hypertrophic growth in rat neonatal cardiomyocytes, and in vivo pressure overload-induced cardiac hypertrophy in mice. These findings on important roles of native TRPC3 channels are strikingly consistent with previous genetic studies. Thus, the TRPC3-selective inhibitor Pyr3 is a powerful tool to study in vivo function of TRPC3, suggesting a pharmaceutical potential of Pyr3 in treatments of TRPC3-related diseases such as cardiac hypertrophy.