Comparative analysis of the reactive oxygen species-producing enzymatic activity of Arabidopsis NADPH oxidases.

Reactive oxygen species (ROS) produced by NADPH oxidases, called respiratory burst oxidase homologs (Rbohs), play crucial roles in development as well as biotic and abiotic stress responses in plants. Arabidopsis has 10 Rboh genes, AtRbohA to AtRbohJ. Five AtRbohs (AtRbohC, -D, -F, -H and -J) are synergistically activated by Ca2+ -binding and protein phosphorylation to produce ROS that play various roles in planta, although the activities of the other Rbohs remain unknown. With a heterologous expression system, we found a range of ROS-producing activity among the AtRbohs with differences up to 100 times, indicating that the required amounts of ROS are different in each situation where AtRbohs act. To specify the functions of AtRbohs involved in cell growth, we focused on AtRbohC, -H and -J, which are involved in tip growth of root hairs or pollen tubes. Ectopic expression of the root hair factor AtRbohC/ROOT HAIR DEFECTIVE 2 (RHD2) in pollen tubes restored the atrbohH atrbohJ defects in tip growth of pollen tubes. However, expression of AtRbohH or -J in root hairs did not complement the tip growth defect in the atrbohC/rhd2 mutant. Our data indicate that Rbohs possess different ranges of enzymatic activity, and that some Rbohs have evolved to carry specific functions in cell growth.

[Successful Treatment of Non-Small Cell Lung Cancer with Afatinib after Gefitinib-Induced Hepatotoxicity].

A 71-year-old man was referred to our hospital for treatment of non-small-cell lung cancer (adenocarcinoma of the lungs, with multiple bone metastases, cT1bN0M1b, harboring an EGFR mutation [exon 19 deletion]). Gefitinib was administered as daily oral doses of 250 mg. After 18 days, gefitinib was discontinued because of hepatotoxicity (Grade 3). Forty days later, afatinib was administered as daily oral doses of 40 mg. Within 1 year, the primary tumor and bone metastases achieved a partial response without hepatotoxicity. We suggest that afatinib is an effective and well-tolerated treatment option for patients with hepatotoxicity under gefitinib treatment.

A low temperature-inducible protein AtSRC2 enhances the ROS-producing activity of NADPH oxidase AtRbohF.

Reactive oxygen species (ROS) produced by NADPH oxidases play critical roles in plant environmental responses. Arabidopsis thaliana NADPH oxidase AtRbohF-mediated ROS-production is involved in abiotic stress responses. Because overproduction of ROS is highly toxic to cells, the activity of AtRbohF needs to be tightly regulated in response to diverse stimuli. The ROS-producing activity of AtRbohF is activated by Ca(2+) and protein phosphorylation, but other regulatory factors for AtRbohF are mostly unknown. In this study, we screened for proteins that interact with the N-terminal cytosolic region of AtRbohF by a yeast two-hybrid screen, and isolated AtSRC2, an A. thaliana homolog of SRC2 (soybean gene regulated by cold-2). A co-immunoprecipitation assay revealed that AtSRC2 interacts with the N-terminal region of AtRbohF in plant cells. Intracellular localization of GFP-tagged AtSRC2 was partially overlapped with that of GFP-tagged AtRbohF at the cell periphery. Co-expression of AtSRC2 enhanced the Ca(2+)-dependent ROS-producing activity of AtRbohF in HEK293T cells, but did not affect its phosphorylation-dependent activation. Low-temperature treatment induced expression of the AtSRC2 gene in Arabidopsis roots in proportion to levels of ROS production that was partially dependent on AtRbohF. Our findings suggest that AtSRC2 is a novel activator of Ca(2+)-dependent AtRbohF-mediated ROS production and may play a role in cold responses.

Myoid hamartoma of the breast that proved difficult to diagnose: a case report.

Myoid hamartomas of the breast are extremely rare breast lesions, with a poorly understood pathogenesis. We describe the case of a 38-year-old premenopausal woman who presenting with a mass in the left breast. Mammography revealed an oval mass that was partly indistinct, and ultrasonography showed a hypoechoic mass with a slightly irregular margin. Bilateral breast dynamic magnetic resonance imaging was performed for a more detailed evaluation. The images showed rapid initial enhancement and a microlobulated margin. Because the suspicion of malignancy was strong at that time, core needle biopsy was performed. Histologically, the tumor was identified as fibroadenoma. A case of myoid hamartoma of the breast that proved difficult to diagnose is reported, and discussed with reference to the literature.

[A case of breast cancer with brain metastases responding to paclitaxel and capecitabine therapy].

A 31-year-old woman was referred to our hospital for back pain. After a close investigation, she was diagnosed with multiple bone, liver and brain metastases of breast cancer. She was administered a combination therapy of paclitaxel and capecitabine. Capecitabine was administered orally at 628mg/m / 2 twice daily on days 1-21, and paclitaxel 80 mg/m2 was injected on days 1, 8, and 15. Both drug courses were repeated every 28 days. After 5 courses of treatment, the level of tumor markers and all metastases were reduced. The combination treatment of paclitaxel and capecitabine is considered to be effective for metastatic breast cancer with brain metastases.

[Administration of nab-paclitaxel for 9 metastatic breast cancer patients].

Nab-paclitaxel was administered to 9 patients with refractory advanced or recurrent breast cancer from 1 to 8 times(median 4)triweekly. The median cumulative dose was 775mg/m2(range 260-2, 000), and the median delivered dose intensity was 66. 7mg/m2/week(range 58. 3-86. 7). The response to treatment was CR in one patient, PR in 2 patients, SD in one patient, and PD in 4 patients. In one patient, treatment had to be suspended because of grade 3 peripheral neuropathy. The clinical benefit was 33%. All 4 PD patients were administered other salvage treatments and are alive. Adverse events included 6 case of neutropenia(grade 3-4 in 4 cases), grade 3 AST/ALT elevations in one patient, grade 3 myalgia in one patient. No case of febrile neutropenia was seen. All reactions were under control except for one patient with grade 3 peripheral neuropathy. Concurrent trastuzumab administration was safe also. In conclusion, nab-paclitaxel could be administered safely, and may contribute to the treatment of refractory advanced or recurrent breast cancer.

[A case of effective lapatinib/capecitabine therapy for HER2-positive breast cancer with multiple brain metastases].

A 63-year-old woman was suffering from HER2-positive and hormone receptor-negative breast cancer with bone metastasis. She received 16 cycles of paclitaxel(PTX 80mg/m2)plus trastuzumab(TRA 2mg/kg)on a 7-day cycle, and zoledronic acid(ZOL 4mg/body every 28 days), resulting in a near clinical complete response(cCR). Two years later, the patient complained of dizziness and nausea, and magnetic resonance imaging revealed multiple brain metastases. The prior treatments with PTX and TRA were changed to lapatinib(LAP)(orally at 1, 250mg/day every day)and capecitabine(CAP)(orally at 2, 000mg/m2 every day for 2 weeks, followed by a 1-week rest interval as 1 cycle)because of the multiple brain metastases. After 4 cycles of treatment, the number of brain lesions and the tumor sizes were significantly reduced. After 7 cycles, however, magnetic resonance imaging revealed the deterioration of some brain lesions. After whole-brain irradiation(30 Gy in 10 fractions)was added to the treatment, the outcome was near cCR. In conclusion, combination therapy of Lap and Cap may be an effective treatment option for brain metastasis of HER2-positive breast cancer.

[A case of effective combination therapy with docetaxel, cyclophosphamide and trastuzumab as primary systemic therapy for locally advanced HER2-positive breast cancer].

We experienced a case of locally advanced breast cancer achieving a significant improvement by using a combination of docetaxel(DOC), cyclophosphamide(CPA)and trastuzumab as a primary systemic therapy.The patient was a 54-year-old woman suffering from a right breast mass, who was referred to our hospital and diagnosed with HER2-positive breast cancer with subclavicular lymph nodes metastases.The combination therapy of DOC(75 mg/m / 2), CPA(600 mg/m2)and trastuzumab(loading dose 8 mg/kg, then 6 mg/kg)for 6 courses at q3 week intervals, was started as the primary systemic therapy. After 6 courses of treatment, a right modified radical mastectomy was performed.There were a little breast cancer cells in the breast, and no axillary lymph node metastases.The combination chemotherapeutic regime with DOC, CPA and trastuzumab seems to be useful for treatment of HER2-positive breast cancer.

Docetaxel, cyclophosphamide, and trastuzumab as neoadjuvant chemotherapy for HER2-positive primary breast cancer.

BACKGROUND: The standard primary systemic therapy for human epidermal growth factor receptor 2-positive (HER2+) breast cancer is anthracyclines and/or taxanes combined with trastuzumab, which demonstrates a high pathological complete response (pCR). A pCR is a predictive marker of prognosis. However, results slightly differ, depending on the hormone receptor status. The efficacy and tolerability of docetaxel, cyclophosphamide, and trastuzumab (HER-TC) as neoadjuvant chemotherapy (NAC) remain unclear. We performed a prospective multicenter study of HER-TC NAC for HER2+ primary breast cancer. METHODS: Eligible patients had a clinical diagnosis of HER2+ invasive breast cancer greater than 1 cm but less than 7 cm and a tumor stage of N0 or N1. T hey were diagnosed between July 2011 and February 2014. For NAC, four cycles of HER-TC (6 mg/kg loading dose, 8 mg/kg, 75, and 600 mg/m2) were administered intravenously every 3 weeks. We investigated the pCR of the primary breast tumors. A pCR was defined as no histological evidence of invasive carcinoma or the appearance of only ductal carcinoma in situ. RESULTS: We enrolled 42 patients. The completion rate for four cycles of HER-TC was 97.6 % (41/42 patients). The overall pCR rate was 43.9 % (18/41 patients). The pCR rate for patients with the luminal HER2 subtype [estrogen receptor (ER)-positive+, HER2+] and the HER2-enriched subtype (ER-, HER2+) was 40.0 % (8/20 patients) and 47.6 % (10/21 patients), respectively. A pCR was achieved with nearly the same probability for each subtype. CONCLUSIONS: Four cycles of HER-TC may be a NAC option for HER2-positive breast cancer.

Which patients require or can skip biopsy for breast clustered microcysts? Predictive findings of breast cancer and mucocele-like tumor.

BACKGROUND: In order to determine the patients who require biopsy for breast clustered microcysts, we retrospectively investigated cases of clustered microcysts that were detected by ultrasonography (US) examinations. METHODS: We investigated 52 patients in whom clustered microcysts were detected by US and who underwent biopsy at our institution between January 2011 and March 2014. These patients were divided into two groups according to histopathological findings: those with invasive carcinoma (IC), ductal carcinoma in situ (DCIS), or mucocele-like tumor (MLT) (Group 1), and those with benign lesions (Group 2). Mammography (MG) and magnetic resonance imaging (MRI) findings were assessed in these two groups. RESULTS: There were 12 patients in Group 1 (IC:2, DCIS:6, and MLT:4) and 40 patients in Group 2. In 51 of the 52 patients, MG was performed before biopsy. Since 11 of 12 patients in Group 1 exhibited MG findings (mass, focal asymmetry, coarse heterogeneous calcifications and fine pleomorphic calcifications) at the same site as the clustered microcysts, and that MG findings had a significant probability of detecting clustered microcysts categorized into Group 1 with a sensitivity of 91.7 % and negative predictive value (NPV) of 96.8 % (p < 0.001). In contrast, MRI was performed in 39 of the 52 patients, and only 3 of the 9 Group 1 patients (33.3 %) for whom MRI was performed were detected as MRI Category 4 or 5 enhancement. CONCLUSION: The patients who exhibit clustered microcysts with MG findings (mass, focal asymmetry, coarse heterogeneous calcifications and fine pleomorphic calcifications) appear to require biopsy.

The CBL-interacting protein kinase CIPK26 is a novel interactor of Arabidopsis NADPH oxidase AtRbohF that negatively modulates its ROS-producing activity in a heterologous expression system.

The plant NADPH oxidases, known as respiratory burst oxidase homologues (Rbohs), play an indispensable role in a wide array of cellular and developmental processes. Arabidopsis thaliana RbohF (AtRbohF)-mediated production of reactive oxygen species (ROS) is involved in biotic and abiotic stress responses. Because of the toxicity of excess amount of ROS, the ROS-producing activity of Rbohs is speculated to be negatively regulated. However, its mechanism is mostly unknown to date. Here, we report the identification of calcineurin B-like protein-interacting protein kinase 26 (CIPK26) as a novel regulatory factor of AtRbohF. We isolated CIPK26 as an AtRbohF-interacting partner by a yeast two-hybrid screen. Our co-immunoprecipitation assay revealed that the CIPK26 protein interacts with the N-terminal region of AtRbohF in Nicotiana benthamiana cell extracts. The fluorescence of both GFP-tagged CIPK26 and AtRbohF was predominantly observed at the cell periphery. We also showed that co-expression of CIPK26 decreases the ROS-producing activity of AtRbohF in HEK293T cells. Together, these results suggest that the direct binding of CIPK26 to AtRbohF negatively modulates ROS production and play a role in the regulation of ROS signalling in plants.

Quality control of photosystem II: lipid peroxidation accelerates photoinhibition under excessive illumination.

Environmental stresses lower the efficiency of photosynthesis and sometimes cause irreversible damage to plant functions. When spinach thylakoids and Photosystem II membranes were illuminated with excessive visible light (100-1,000 µmol photons m(-1) s(-1)) for 10 min at either 20°C or 30°C, the optimum quantum yield of Photosystem II decreased as the light intensity and temperature increased. Reactive oxygen species and endogenous cationic radicals produced through a photochemical reaction at and/or near the reaction center have been implicated in the damage to the D1 protein. Here we present evidence that lipid peroxidation induced by the illumination is involved in the damage to the D1 protein and the subunits of the light-harvesting complex of Photosystem II. This is reasoned from the results that considerable lipid peroxidation occurred in the thylakoids in the light, and that lipoxygenase externally added in the dark induced inhibition of Photosystem II activity in the thylakoids, production of singlet oxygen, which was monitored by electron paramagnetic resonance spin trapping, and damage to the D1 protein, in parallel with lipid peroxidation. Modification of the subunits of the light-harvesting complex of Photosystem II by malondialdehyde as well as oxidation of the subunits was also observed. We suggest that mainly singlet oxygen formed through lipid peroxidation under light stress participates in damaging the Photosystem II subunits.