Red-Beet Betalain Pigments Inhibit Amyloid-ß Aggregation and Toxicity in Amyloid-ß Expressing Caenorhabditis elegans.

Betalain pigments are mainly produced by plants belonging to the order of Caryophyllales. Betalains exhibit strong antioxidant activity and responds to environmental stimuli and stress in plants. Recent reports of antioxidant, anti-inflammatory and anti-cancer properties of betalain pigments have piqued interest in understanding their biological functions. We investigated the effects of betalain pigments (betanin and isobetanin) derived from red-beet on amyloid-ß (Aß) aggregation, which causes Alzheimer's disease. Non-specific inhibition of Aß aggregation against Aß40 and Aß42 by red-beet betalain pigments, in vitro was demonstrated using the thioflavin t fluorescence assay, circular dichroism spectroscopy analysis, transmission electron microscopy and nuclear magnetic resonance (NMR) analysis. Furthermore, we examined the ability of red-beet betalain pigments to interfere with Aß toxicity by using the transgenic Caenorhabditis elegans model, which expresses the human Aß42 protein intracellularly within the body wall muscle. It responds to Aß-toxicity with paralysis and treatment with 50 µM red-beet betalain pigments significantly delayed the paralysis of C. elegans. These results suggest that betalain pigments reduce Aß-induced toxicity.

First-line sorafenib sequential therapy and liver disease etiology for unresectable hepatocellular carcinoma using inverse probability weighting: A multicenter retrospective study.

BACKGROUND AND AIMS: Sequential therapy with molecular-targeted agents (MTAs) is considered effective for unresectable hepatocellular carcinoma (HCC) patients. This study purposed to evaluate the efficacy of sequential therapy with sorafenib (SORA) as a first-line therapy and to investigate the therapeutic impact of SORA in nonalcoholic fatty liver disease (NAFLD) or nonalcoholic steato hepatitis (NASH)-related HCC. METHODS: We evaluated 504 HCC patients treated with SORA (Study-1). The times of administration for sorafenib from 2009 to 2015, 2016 to 2017, and 2018 and later were defined as the early-, mid-, and late-term periods, respectively. Among them, 180 HCC patients treated with SORA in addition to MTAs in the mid- and late-term periods were divided into groups based on disease etiology (NAFLD or NASH [n = 37] and viral or alcohol [n = 143]), and outcomes were compared after inverse probability weighting (IPW) (Study-2). RESULTS: Overall survival (OS) of HCC patients who received sequential MTA therapy after first-line SORA was significantly longer. The median survival times (MST) were 12.6 versus 17.6 versus 17.4 months in the early-term group, mid-term group, and the later-time group (early vs. mid, p = 0.014, early vs. later. p = 0.045), respectively. (Study-1). In Study-2, there was no significant differences in OS between the Virus/alcohol group and the NAFLD/NASH group in patients who received sequential therapy (MST was 23.4 and 27.0 months p = 0.173, respectively). The NAFLD or NASH, female sex, albumin-bilirubin (ALBI) grade 2b, and major Vp (Vp3/Vp4) were significant factors for OS treated with SORA. CONCLUSIONS: Sequential therapy with SORA as the first-line treatment improved the prognosis of unresectable HCC patients and was effective regardless of HCC etiology.

Lenvatinib versus sorafenib in first-line treatment of unresectable hepatocellular carcinoma: An inverse probability of treatment weighting analysis.

PURPOSE: Data from common clinical practice were used to generate balanced cohorts of patients receiving either sorafenib or lenvatinib, for unresectable hepatocellular carcinoma, with the final aim to investigate their declared equivalence. METHODS: Clinical features of lenvatinib and sorafenib patients were balanced through inverse probability of treatment weighting (IPTW) methodology, which weights patients' characteristics and measured outcomes of each patient in both treatment arms. Overall survival was the primary endpoint and occurrence of adverse events was the secondary. RESULTS: The analysis included 385 patients who received lenvatinib, and 555 patients who received sorafenib. In the unadjusted cohort, lenvatinib did not show a survival advantage over sorafenib (HR: 0.85, 95% CI 0.70-1.02). After IPTW adjustment, lenvatinib still not returned a survival advantage over sorafenib (HR: 0.82, 95% CI: 0.62-1.07) even in presence of balanced baseline characteristics. Lenvatinib provided longer survival than sorafenib in patients previously submitted to TACE (HR: 0.69), with PS of 0 (HR: 0.73) or without extrahepatic disease (HR: 0.69). CONCLUSION: Present results confirmed randomized controlled trial in the real-life setting, but also suggests that in earlier stages some benefit can be expected.

Efficacy of a Glass Membrane Emulsification Device to Form Mixture of Cisplatin Powder with Lipiodol on Transarterial Therapy for Hepatocellular Carcinoma.

PURPOSE: To examine physiochemical characteristics and drug release properties of cisplatin powder and lipiodol mixtures formed by a glass membrane emulsification device compared with a 3-way stopcock. MATERIALS AND METHODS: Seven different types of mixtures were evaluated: cisplatin powder and lipiodol directly mixed (suspension), complete cisplatin solution and lipiodol mixed by a 3-way stopcock or the device (emulsion), incomplete cisplatin solution and lipiodol mixed by a 3-way stopcock or the device (solid-in-water emulsion), and contrast material and cisplatin suspension mixed by a 3-way stopcock or the device (solid-in-oil emulsion). RESULT: The percentages of water-in-oil were 98.08 ± 0.27% in the emulsion formed by the device, while 70.3 ± 4.63% in the emulsion formed by a 3-way stopcock (P = 0.037). Solid-in-water and solid-in-oil emulsions formed by the device showed 98.09 ± 0.38% and 98.70 ± 0.40% of water-in-oil, respectively, whereas both solid-in-water and solid-in-oil emulsions formed by a 3-way stopcock showed 0.00%. Homogenous droplet sizes were shown by using the device. The half release times of cisplatin in the emulsions formed by the device were 197 ± 19, 244 ± 24 and 478 ± 52 min, respectively, which were significantly longer than the emulsion formed by a 3-way stopcock of 8 ± 8 min (P = 0.046-0.050). Suspension showed the longest release time; however, the viscosity was lowest. CONCLUSION: The glass membrane emulsification device formed almost 100% water-in-oil, whereas 3-way stopcock produced 100% oil-in-water when incomplete solution or suspension was mixed. Slower cisplatin release was shown in the emulsions formed by the device.

Controlling Nutritional Status (CONUT) Score is Associated with Overall Survival in Patients with Unresectable Hepatocellular Carcinoma Treated with Lenvatinib: A Multicenter Cohort Study.

We aimed to investigate the impact of the controlling nutritional status (CONUT) score, an immuno-nutritional biomarker, on the prognosis of patients with hepatocellular carcinoma (HCC) treated with lenvatinib (LEN). This retrospective study enrolled 164 patients with HCC and treated with LEN (median age 73 years, Barcelona Clinic Liver Cancer (BCLC) stage B/C 93/71). Factors associated with overall survival (OS) were evaluated using multivariate and decision tree analyses. OS was calculated using the Kaplan-Meier method and analyzed using the log-rank test. Independent factors for OS were albumin-bilirubin grade 1, BCLC stage B, and CONUT score <5 (hazard ratio (HR) 2.9, 95% confidence interval (CI) 1.58-5.31, p < 0.001). The CONUT score was the most important variable for OS, with OS rates of 70.0% and 29.0% in the low and high CONUT groups, respectively. Additionally, the median survival time was longer in the low CONUT group than in the high CONUT group (median survival time not reached vs. 11.3 months, p < 0.001). The CONUT score was the most important prognostic variable, rather than albumin-bilirubin grade and BCLC stage, in patients with HCC treated with LEN. Accordingly, immuno-nutritional status may be an important factor in the management of patients with HCC treated with LEN.

Epirubicin is More Effective than Miriplatin in Balloon-Occluded Transcatheter Arterial Chemoembolization for Hepatocellular Carcinoma.

BACKGROUND: Transcatheter arterial chemoembolization (TACE) is a standard therapy used in the treatment of intermediate hepatocellular carcinoma (HCC). Recently, balloon-occluded TACE (B-TACE) has been developed. PURPOSE: This study aimed to clarify the effects of B-TACE in patients with HCC, with a focus on which drug is suitable to suspend in Lipiodol for B-TACE. METHODS: We retrospectively evaluated 35 patients with HCC treated with B-TACE. Factors associated with enhanced time to progression (TTP) after B-TACE were evaluated using univariate and multivariate analyses. RESULTS: A total of 35 patients with HCC (40 nodules) were treated with B-TACE between June 2013 and August 2016. Epirubicin was used in 25 nodules and miriplatin was used in 15 nodules. Epirubicin (15.1 months) was significantly better than miriplatin (3.2 months) in prolonging the local TTP after B-TACE (p = 0.0293). Epirubicin showed a positive tendency in TE4 (100% tumor necrosis) rate when compared with miriplatin (p = 0.058). Achievement of TE4 was the only significant factor associated with better TTP after B-TACE. Epirubicin- and TACE-naïve statuses were significant factors in achieving TE4 with B-TACE. CONCLUSION: To enhance the TTP with B-TACE, TE4 should be achieved. Epirubicin is a more optimal anticancer drug (as a Lipiodol suspension) than miriplatin for achieving TE4 with B-TACE.

Pigment Epithelium-Derived Factor (PEDF) Prevents Hepatic Fat Storage, Inflammation, and Fibrosis in Dietary Steatohepatitis of Mice.

BACKGROUND AND AIMS: Pigment epithelium-derived factor (PEDF) has been shown to be a potent inhibitor of inflammation through its anti-oxidative property. Since oxidative response is considered to play the pivotal role of the development and progression of nonalcoholic steatohepatitis (NASH), it is conceivable that PEDF may play a protective role against NASH. In this study, we examined whether administration of PEDF slowed the progression of NASH in mice models. METHODS: Mice were fed methionine- and choline-deficient (MCD) diet with or without intramuscular administration of adenovirus-expressing PEDF (Ad-PEDF). Effects of PEDF administration on NASH were histologically and biochemically evaluated. RESULTS: Administration of Ad-PEDF significantly decreased hepatic fat storage as well as serum levels of ALT in MCD diet-fed mice. Dihydroethidium staining showed that MCD diet-triggered oxidative stress was reduced in the liver of Ad-PEDF-administered mice compared to that of PBS- or Ad-LacZ-administered mice. Activation of Kupffer cells and hepatic fibrosis was also inhibited by Ad-PEDF administration. Quantitative real-time RT-PCR revealed that MCD diet up-regulated expressions of TNF-α, IL-1ß, IL-6, TGF-ß, collagen-1, and collagen-3 mRNA, which were also attenuated with Ad-PEDF administration, whereas MCD diet-induced down-regulation of expressions of PPAR-γ mRNA was restored with Ad-PEDF administration. Furthermore, immunoblotting analysis showed that MCD diet-induced up-regulation of NADPH oxidase components was significantly decreased in Ad-PEDF-administered mice. CONCLUSIONS: The present results demonstrated for the first time that PEDF could slow the development and progression of steatohepatitis through the suppression of steatosis and inflammatory response in MCD diet-fed mice. Our study suggests that PEDF supplementation may be a novel therapeutic strategy for the treatment of NASH.

Alternative treatments in advanced hepatocellular carcinoma patients with progressive disease after sorafenib treatment: a prospective multicenter cohort study.

Sorafenib is an oral multikinase inhibitor that has been approved to treat advanced hepatocellular carcinoma (HCC), though it is unclear how much benefit advanced HCC patients with progressive disease (PD) derive from sorafenib treatment. This study aimed to assess survival risk factors and evaluate therapeutic strategies for advanced HCC patients with PD after sorafenib treatment. We analyzed the clinical data and treatment outcomes for 315 consecutive advanced HCC patients treated with sorafenib. Univariate analyses of overall survival identified therapeutic effect as an independent risk factor in all patients. Among all patients, 141 developed PD. Of those, 58 (41%) were treated with sorafenib monotherapy, 70 (50%) with agents other than sorafenib, and 13 (9%) were not treated at all. The median survival time was 6.1 months for PD patients with sorafenib monotherapy and 12.2 months for those administered alternative treatments (p < 0.0001). Our results indicated that sorafenib treatment may have negative long-term therapeutic effects in advanced HCC patients with PD, and that alternative treatments should be considered for these patients after sorafenib administration.

Hepatic arterial infusion chemoembolization therapy for advanced hepatocellular carcinoma: multicenter phase II study.

PURPOSE: Portal vein tumor thrombosis is a critical complication in patients with hepatocellular carcinoma (HCC). This prospective multicenter trial assessed the efficacy of hepatic arterial infusion chemoembolization therapy with cisplatin suspended in lipiodol combined with 5-fluorouracil for HCC patients with portal vein tumor thrombosis. METHODS: We enrolled 52 HCC patients with portal vein tumor thrombosis. They received hepatic arterial infusion chemoembolization therapy with cisplatin suspension in lipiodol and 5-fluorouracil. The primary efficacy endpoint was progression-free survival (PFS), while the secondary endpoints were overall survival (OS), tumor response rate, safety, and tolerability. Independent factors for survival were also evaluated. RESULTS: The median PFS and OS were 8.6 and 27.0 months, respectively. Ten patients showed complete response, while 29 had partial response (response rate, 75.0 %). The median survival time of 10 patients with complete response and 29 with partial response was 32 months, while that of 15 patients with partial response who later showed disappearance of HCC following additional therapies was 50 months. Multivariate analysis identified response to treatment and disappearance of viable HCC as independent predictors of survival. The treatment was well tolerated, and the only encountered Grade 3 toxicities were thrombocytopenia and hyperbilirubinemia. CONCLUSIONS: Hepatic arterial infusion chemoembolization therapy with cisplatin suspension in lipiodol combined with 5-fluorouracil is effective treatment for unresectable HCC with portal vein tumor thrombosis.

Sorafenib for the treatment of advanced hepatocellular carcinoma with extrahepatic metastasis: a prospective multicenter cohort study.

Sorafenib, an oral multikinase inhibitor, is approved for advanced hepatocellular carcinoma (HCC) treatment. However, its therapeutic effect in advanced HCC patients with extrahepatic metastasis remains uncertain. This study aimed to prospectively assess the efficacy, safety, and survival risk factors and evaluate the prognostic impact of sorafenib treatment in advanced HCC patients with or without extrahepatic metastasis. Between May 2009 and March 2014, 312 consecutive advanced HCC patients who received sorafenib were enrolled in this study. We evaluated their characteristics and compared the clinical outcomes of those with and without extrahepatic metastasis. Of the enrolled patients, 245 (81%) received sorafenib treatment for more than 1 month, with a median duration of 3.6 months. Eighteen patients demonstrated partial response to sorafenib therapy, 127 had stable disease, and 134 had progressive disease at the first radiologic assessment. The median survival time (MST) and progression-free survival (PFS) were 10.3 and 3.6 months, respectively. Multivariate analysis identified gender, Child-Pugh class, baseline serum des-gamma-carboxy prothrombin level, and treatment duration as independent risk factors for survival. Extrahepatic metastasis was detected in 178 patients. However, the MST, PFS, and therapeutic effect were comparable between patients with and without extrahepatic metastasis. The independent risk factors for decreased overall survival in patients with extrahepatic metastasis were similar to those affecting all patients. Our results indicated that sorafenib could be administered for hepatic reserve and as long-term treatment for advanced HCC patients regardless of their extrahepatic metastasis status.

Efficacy, safety, and survival factors for sorafenib treatment in Japanese patients with advanced hepatocellular carcinoma.

BACKGROUND: Sorafenib, an oral multikinase inhibitor, was approved for the treatment of advanced hepatocellular carcinoma (HCC), but has not been adequately evaluated for safety and effectiveness in Japanese patients with advanced HCC. AIMS: The purpose of this study was to prospectively assess the efficacy, safety, and risk factors for survival in patients with advanced HCC treated with sorafenib. METHODS: Between May 2009 and December 2010, 96 Japanese patients with advanced HCC (76 male, 20 female, mean age: 70.4 years) were treated with sorafenib. Eighty-eight patients had Child-Pugh class A, and 8 patients had Child-Pugh class B liver cirrhosis. Barcelona Clinic Liver Cancer stage B and C were found in 64 and 32 patients, respectively. RESULTS: Twelve patients demonstrated partial response to sorafenib therapy, 43 patients had stable disease, and 33 patients had progressive disease at the first radiologic assessment. The most frequent adverse events leading to discontinuation of sorafenib treatment were liver dysfunction (n = 8), hand-foot skin reaction (n = 7), and diarrhea (n = 4). The median survival time and time to progression were 11.6 and 3.2 months, respectively. By multivariate analysis, des-γ-carboxy prothrombin serum levels and duration of treatment were identified as independent risk factors for survival. CONCLUSIONS: This study showed that sorafenib was safe and useful in Japanese patients with advanced HCC. In addition, this study demonstrated that sorafenib should be administered as a long-term treatment for advanced HCC regardless of therapeutic effect and dosage.

New lipoic acid derivative drug sodium zinc dihydrolipoylhistidinate prevents cardiac dysfunction in an isolated perfused rat heart model.

OBJECTIVE: Myocardial ischemia/reperfusion injury is a life-limiting condition. Reactive oxygen species are released upon reperfusion, resulting in damage to myocardial cells. Accordingly, antioxidant drugs have been shown to protect the myocardium against ischemia/reperfusion injury. The purpose of the present study was to determine the cardioprotective effects of the new lipoic acid-derivative drug sodium zinc dihydrolipoylhistidinate in a global ischemia isolated perfused rat heart model. DESIGN: Animals were randomly divided into five groups: 1) normal group, 2) control ischemia/reperfusion group, 3) high-dose sodium zinc dihydrolipoylhistidinate (1 ng/mL) plus ischemia/reperfusion group, 4) medium-dose sodium zinc dihydrolipoylhistidinate (0.1 ng/mL) plus ischemia/reperfusion group, or 5) low-dose sodium zinc dihydrolipoylhistidinate (0.01 ng/mL) plus ischemia/reperfusion group. SETTING: University medical center research laboratory. SUBJECTS: Male Sprague-Dawley rats weighing 250-300 g. MEASUREMENTS AND MAIN RESULTS: Hearts underwent ischemia/reperfusion after isolation with or without sodium zinc dihydrolipoylhistidinate treatment. We then conducted cardiac histopathology and transmission electron microscopy analyses and assessed cardiac function. In addition, we examined the effects of sodium zinc dihydrolipoylhistidinate on ischemia/reperfusion-induced mitochondrial dysfunction. We found that cardiac dysfunction and mitochondrial damage were significantly reduced after reperfusion by sodium zinc dihydrolipoylhistidinate treatment. However, only rats treated with high-dose sodium zinc dihydrolipoylhistidinate showed improved cardiac function. Furthermore, treatment with sodium zinc dihydrolipoylhistidinate significantly improved mitochondrial function in vitro. CONCLUSIONS: These findings suggest that sodium zinc dihydrolipoylhistidinate attenuates ischemia/reperfusion-induced myocardial dysfunction in rats. Furthermore, sodium zinc dihydrolipoylhistidinate exerted cardioprotective effects via preservation of mitochondrial function. Taken together, our results strongly support the potential therapeutic role of sodium zinc dihydrolipoylhistidinate in the treatment of ischemia/reperfusion injury.

The impact of oxidative stress levels on the clinical effectiveness of sivelestat in treating acute lung injury: an electron spin resonance study.

BACKGROUND: Sivelestat, a neutrophil elastase inhibitor, has been used to treat acute lung injury (ALI) with varying levels of clinical success. Variable baseline levels of oxidative stress in patients with ALI have been proposed as one explanation for inconsistent results. METHODS: Using a bedside electron spin resonance spectrometer, we evaluated electron spin resonance signal intensities of serum ascorbyl free radicals supplemented with dimethyl sulfoxide (AFR/DMSO) in patients with ALI. RESULTS: We found a positive correlation between AFR/DMSO and ascorbate levels, suggesting that serum AFR/DMSO measurements may serve as a surrogate for real-time assessments of oxidative stress. Levels of AFR/DMSO in patients with ALI were significantly lower than those found in healthy controls. Stratified analyses revealed that baseline AFR/DMSO levels were significantly lower in patients with ALI who failed to respond to sivelestat compared with those who did respond. CONCLUSIONS: Our results suggest that the clinical efficacy of sivelestat is dependent on baseline oxidative stress levels.

Epigallocatechin-3-gallate improves nonalcoholic steatohepatitis model mice expressing nuclear sterol regulatory element binding protein-1c in adipose tissue.

We examined whether or not epigallocatechin-3-gallate (EGCG) improves liver injury of nonalcoholic steatohepatitis (NASH) model mice expressing nuclear sterol regulatory element-binding protein 1c (nSREBP-1c) in adipose tissue. nSREBP-1c transgenic C57BL6 mice aged 30 weeks were divided into group 1 (no treatment), group 2 (ascorbic acid alone), group 3 (ascorbic acid and 0.05% EGCG), and group 4 (ascorbic acid and 0.1% EGCG). At 42 weeks, we performed measurement of liver weight to body weight, biochemical assays, morphometry of liver specimens, immunohistochemistry for 8-hydro-2'-deoxyguanosine (8-OhdG), and Western blotting for insulin and TNF-alpha signalings. Ratio of liver weight to body weight in the high dose EGCG-treated group (group 4) was significantly lower than those of groups 1 and 2 (p<0.05 and <0.01, respectively). Blood ALT, glucose, total cholesterol, and triglyceride levels of group 4 were significantly low compared with those of the EGCG-non-treated group (groups 1 and 2) (p<0.05, respectively). The degrees of steatosis, inflammation, ballooning hepatocytes and Mallory-Denk bodies in group 4 significantly improved compared with those in other groups (p<0.05, respectively). The 8-OhdG immunolocalization in liver tissues of the group 4 obviously decreased compared with those of groups 2 and 3. For Western blotting, the expressions of insulin receptor substrate-1 (IRS-1) and phosphorylated IRS-1 (pIRS-1) in liver tissues of group 4 increased compared with those of groups 2 and 3. On the other hand, the expressions of pAkt, pIKKbeta and pNF-kappaB decreased compared with those of groups 2 and 3. From these results, EGCG reduces inflammation, insulin resistance and oxidative stress, and suppresses liver injury in nSREBP-1c transgenic mice.

The Wilson disease protein ATP7B resides in the late endosomes with Rab7 and the Niemann-Pick C1 protein.

Wilson disease is a genetic disorder characterized by the accumulation of copper in the body due to a defect of biliary copper excretion. Although the Wilson disease gene has been cloned, the cellular localization of the gene product (ATP7B) has not been fully clarified. Therefore, the precise physiological action of ATP7B is still unknown. We examined the distribution of ATP7B using an anti-ATP7B antibody, green fluorescent protein (GFP)-ATP7B (GFP-ATP7B) and ATP7B-DsRed in various cultured cells. Intracellular organelles were visualized by fluorescence microscopy. The distribution of ATP7B was compared with that of Rab7 and Niemann-Pick C1 (NPC1), proteins that localize in the late endosomes. U18666A, which induces the NPC phenotype, was used to modulate the intracellular vesicle traffic. GFP-ATP7B colocalized with various late endosome markers including Rab7 and NPC1 but not with Golgi or lysosome markers. U18666A induced the formation of late endosome-lysosome hybrid organelles, with GFP-ATP7B localized with NPC1 in these structures. We have confirmed that ATP7B is a late endosome-associated membrane protein. ATP7B appears to translocate copper from the cytosol to the late endosomal lumen, thus participating in biliary copper excretion via lysosomes. Thus, defective copper ATPase activity of ATP7B in the late endosomes appears to be the main defect of Wilson disease.

Pistillody, homeotic transformation of stamens into pistil-like structures, caused by nuclear-cytoplasm interaction in wheat.

Homeotic transformation of stamens into pistil-like structures (pistillody) has been observed in a cytoplasmic substitution (alloplasmic) line of wheat (Triticum aestivum L.) cv. Norin 26, which has the cytoplasm of a wild relative species, Aegilops crassa L. On the other hand, an alloplasmic line of wheat cv. Chinese Spring (CS) with Ae. crassa cytoplasm has normal flowers. This is due to the presence in the CS nucleus of a fertility-restoring gene, Rfd1. Deletion mapping analysis revealed that Rfd1 is located on the middle part of the long arm of chromosome 7B. To investigate the function of the Rfd1 gene by a loss-of-function strategy, we produced alloplasmic lines of CS ditelosomic 7BS [(cr)-CSdt7BS] and CS monotelodisomic 7BS [(cr)-CSmd7BS] with the Ae. crassa cytoplasm, and characterized their phenotypes. The line (cr)-CSdt7BS without Rfd1 exhibited pistillody in all florets, and also female sterility. Scanning electron microscopy of the young spikes revealed that the pistillody was induced at an early stage of stamen development. The pistillate stamens often developed incomplete ovule-like structures with integuments instead of tapetum and pollen grains. It is possible that MADS box genes are associated with the induction of pistillody, because the expression of wheat APETALA3 homologue (WAP3) was reduced in the young spikes of (cr)-CSdt7BS. In addition, a histological study indicated that the female sterility in (cr)-CSdt7BS is due to the abnormality of the ovule, which fails to form an inner epidermis and integuments in the chalaza region. The line (cr)-CSmd7BS, hemizygous for Rfd1, showed partial pistillody (51%) and restored female fertility up to 72%. These results suggest that the induction of both pistillody and ovule deficiency caused by the Ae. crassa cytoplasm is inhibited by the Rfd1 gene in a dose-dependent manner.