Few studies have been devoted to NMDARs PXD101 mouse in nonneural tissues, and presence of NMDAR activity in liver has not been defined. Nonetheless, in liver failure, plasma ammonia may induce neurotoxicity via NMDARs in brain, and previously NMDAR antagonists, e. g., MK801 and memantine, improved survival in animals with liver failure. Recently, neurotropic receptor expression

was unexpectedly identified in liver after a period of anoxia, which led us to hypothesize that NMDARs may directly contribute in hepatotoxicity. To develop this possibility, we cultured HuH-7 cells and primary mouse hepatocytes with or without NMDA and acetaminophen (APAP), MK801, and memantine. MTT assays were performed to assess cytotoxicity. Intracellular Ca++ fluxes were measured in hepatocytes with NMDA and NMDAR blockers. Brain and liver tissues were examined for multiple NMDARs by RT-PCR, western, and immunostaining. C57BL/6 mice were used for studies with 500 mg/kg APAP, 2 mg/kg MK801 or Daporinad datasheet 30 mg/kg memantine, Besides mortality, liver injury was evaluated by histology and liver tests. We found NMDAR were expressed in liver at RNA and protein levels. Moreover, HuH-7 cells and mouse hepatocytes were sensitive to NMDA with cytotoxicity as shown by MTT assays. Although APAP-induced cytotoxicity in HuH-7

cells or mouse hepatocytes was not potentiated by simultaneous presence of NMDA, it was abolished when cells were cultured with APAP plus either MK801 or memantine. In mouse hepatocytes, NMDA dose-dependently induced intracellular next Ca++ fluxes. APAP alone did not directly stimulate intracellular Ca++ fluxes, as was expected. By contrast, MK801 and memantine

blocked intracellular Ca++ oscillations. In APAPtreated mice, we observed significant mortality, liver necrosis and liver test abnormalities. When mice treated with APAP were given MK801 or memantine, survival of animals was prolonged and liver histology improved. Conclusions: The NMDARs were expressed in hepatocytes, especially after liver injury, and contributed to APAP-induced hepatotoxicity. Decreases in hepatic injury after blockade of NMDARs by MK801 or memantine indicated further studies of hepatic NMDARs will be helpful. In particular, pathophysiological studies of NMDARs in liver diseases should be relevant for their therapeutic implications. Disclosures: The following people have nothing to disclose: Nicole Pattamanuch, Preeti Viswanathan, Sylvia O. Suadicani, David C. Spray, Sanjeev Gupta Lipid droplets (LDs) are the major cellular storage sites of esterified fatty acids and are the central organelle contributing to hepatic steatosis. The specific machinery orchestrating the breakdown of these structures remains unclear. The goal of this study was to further define the hepatocellular machinery that supports LD metabolism.

3 Such patients may have opioid-induced hyperalgesia, which can occur even after brief exposure to opioids.26 Furthermore, based on the premise that supporting

glia, their receptors, and their secreted mediators may play an important role in neuronal function regulation and so may contribute to migraine,27 Watkins and others have posited a mechanism whereby chronic morphine exposure may modulate glial function, suggesting that the clinical efficacy of opiates for pain control is limited by analgesic tolerance and hyperalgesia.28 A recent study pointed to common cellular mechanisms of opioid-induced desensitization and sensitization mediated through activation of the central glutamatergic system.29 Opioid tolerance and opioid-induced

hyperalgesia are distinct pharmacologic phenomena, but over time, each results in decreased effectiveness of a Histone Methyltransferase inhibitor given opioid dose, leading to dose escalation.25 In opioid-induced hyperalgesia, patients experience pain or increased sensitivity to pain even when serum opioid levels are low. Even at baseline (before the start of the opioid infusion), their pain tolerance is decreased compared to that of opioid-naive patients. In opioid tolerance, the administered dose is no longer effective and the dose must be increased to get the same effect. This reflects desensitization of patients’ antinociceptive pathways from chronic use of opioid medications; no change in pain sensitivity occurs at baseline. How Migraine Medications Work.— Five medications commonly find more used in migraine prophylaxis – topiramate, valproate, propranolol, amitriptyline, and methysergide

– have a common mechanism of action.30 Chronic administration of these 5 migraine preventive medications, Quisqualic acid but not the control L-propranolol, has been shown to reduce CSD in rats, suggesting that suppression of CSD is a common mechanism of action for migraine preventive medications. Ayata et al tested the hypothesis that all of these medications suppress CSD, implicated in migraine attacks. The investigators administered various doses of each of the 5 medications to male Sprague–Dawley rats. Prophylactic efficacy of the 5 medications positively correlated with duration of treatment. Chronic treatment with each of the 5 medications almost completely suppressed CSD after a 17-week course of treatment,30 whereas in human beings, a trial of at least 3 months may be necessary to determine the efficacy of those medications in migraine prevention.3 The study also showed that the efficacy of the medications is dose-dependent.30 Therefore, physicians should begin with a low dose and titrate the dose up when prescribing any of those medications for their patients with migraine.

Sub-G1 cells in flow cytometric histograms were considered apoptotic cells. Analysis of cell cycle distribution and the percentage

of cells in the G1, S, and G2/M phases of the cell cycle were determined using the software FlowJo (Tree Star, Ashland, OR). To detect apoptosis, the Annexin V–FLUOS kit (Roche Diagnostics) was used. Cells were treated for 6, 24, or 48 hours with saffron extract. After washing twice in phosphate-buffered saline, 1 × 106 cells were stained with 100 μL annexin V staining solution, consisting of 20 μL fluorescein isothiocyanate–conjugated RXDX-106 manufacturer annexin V reagent (20 μg/mL), 20 μL isotonic propidium iodide (PI; 50 μg/mL), and 1000 μL of 1 mol/L HEPES buffer, for 15 minutes at room temperature. Cells were analyzed on a FACSCalibur flow cytometer (Becton-Dickinson) using a 488 nm excitation and 530/30 nm band-pass filter for fluorescein detection and a long-pass filter 2P670 nm for PI detection after electronic compensation. Because positive annexin V staining indicates apoptotic STI571 nmr and necrotic cells, PI-positive cells were used to measure late apoptotic cells and necrotic cells, whereas annexin V–positive and

PI-negative cells were counted as early apoptotic cells. Whole-cell lysates were prepared from HepG2 tumor cells. Protein concentration of lysates was determined with a Bio-Rad DC Protein Assay (Bio-Rad Laboratories, Hercules, CA), and 30 μg proteins were loaded onto 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels. The gels were transferred to nitrocellulose membranes before immunodetection processing with anti-phospho-H2AX (Millipore), anti-caspase-3 (Cell Signaling Technology), anti-IκB (Abcam, Cambridge, UK), anti-TNFR1 (Santa Cruz Biotechnology, Santa Cruz, CA), and with secondary antibodies (anti-mouse or anti-rabbit IgG peroxidase conjugated; Pierce, Rockford, IL). Bound antibodies were detected by incubating the blots in West Pico chemiluminescent substrate (Pierce). The level of

however immunoreactivity was measured as peak intensity using an image-capture and analysis system (GeneGnome; Syngene, UK). Hybridization with anti-GAPDH was used to control equal loading and protein quality. SPSS (version 10) statistical program (SPSS Inc., Chicago, IL) was used to carry out a one-way analysis of variance (ANOVA) on our data. When significant differences by ANOVA were detected, analysis of differences between the means of the treated and control groups were performed by using Dunnett’s t test. Other experimental procedures are described in detail in the supporting information. These include animal housekeeping and treatment, in vitro antioxidant properties, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-ending labeling (TUNEL) assay, immunohistochemical analyses, morphology and histopathology analysis, as well as enzyme-linked immunosorbent assay (ELISA).

9+44 days. All patients required prolonged mechanical ventilation (mean duration: 45+28 days), with the need of extracorporal membrane oxygenation (ECMO) in 11 patients. Laboratory findings at the time Selleck Barasertib of ERCP were: bilirubin: 14.9[0.4-18]; (mg/dl, median[range]), GGT: 29[1.3-60.8] (xULN, median[range]), AP: 10.8[1.1-28.0], (xULN, median[range]). Sphincterotomy, extraction of casts/ sludge and dilations of dominant strictures were performed during ERCP. During follow up 26 patients died and 4 patients were transplanted. Number of organ failure and organ replacement therapy were independent risk factors for mortality. Conclusion: Critical reduction of hepatic oxygen delivery may lead to initial

bile duct injury in ICU patients. Vasopressor treatment and sedoanalgesia, hepatic ischemia, as well as translocation of endotoxins and bacteria from the gut may further perpetuate progressing SC-CIP, which

carries a high mortality as a high proportion of these patients rapidly develop liver cirrhosis and liver failure. Disclosures: Harald Hofer – Speaking and Teaching: Janssen, Roche, MSD, Gilead, Abbvie Peter Fickert – Consulting: Falk Foundation, Falk Foundation, Trichostatin A ic50 Falk Foundation, Falk Foundation; Speaking and Teaching: Roche Austria, Gilead Austria, MSD Austria, MERCK Austria, Roche Austria, Gilead Austria, MSD Austria, MERCK Austria, Roche Austria, Gilead Austria, MSD Austria, MERCK Austria, Roche Austria, Gilead Austria, MSD Austria, MERCK Austria Michael Trauner – Advisory Committees or Review Panels: MSD, Janssen, Gilead, Abbvie; Consulting: Phenex; Grant/Research Support: Intercept, Falk Pharma, Albireo; Patent Held/Filed: Med Uni Graz (norUDCA); Speaking and Teaching: Falk Foundation, Roche, Interleukin-3 receptor Gilead The following people have nothing to disclose: Gernot Zollner, Ivo Graziadei Background: dnTGFβRII mice develop high titer AMAs and histologic features characteristic of autoimmune cholangitis, with striking similarities to human PBC. There is increasing interest in the potential use of regulatory T cells (Tregs) as immunotherapy to treat

diseases characterized by loss of tolerance. We have taken advantage of the dnTGFβRII model and, in particular, an ability to induce autoimmune cholangitis in Rag1−/− recipients by adoptive transfer of dysregulated CD8+ T cells from dnT-GFβRII mice. Such adoptively transferred Rag1−/− recipients develop severe portal inflammation with both histologic and cytokine evidence of intense inflammation. Methods: Rag1−/− mice, at four weeks of age, received either CD8+ T cells from dnTGFβRII mice with co-transfer of either Foxp3+ Tregs derived from wild-type and otherwise healthy C57BL/6 mice, or dnTGFβRII mice. Recipient mice were monitored for histology including portal inflammation and intra-lobular biliary cell damage, phenotypic changes in recipient lymphoid populations and local and systemic cytokine production.

Baptista, Emma Moran, Shay Soker Background: Human hepatocytes derived from somatic cells of individuals would be useful in developing cell-based disease models, drug development

and regenerative medicine. Although several types of somatic cells have been reprogrammed to induced pluripotent cells (iPSCs) and then differentiated to hepatocyte-like Adriamycin supplier cells (iHep), the method for generating such cells from renal epithelial cells shed in human urine has not been described systematically. Aim: Reprogram-ming human urinary epithelial cells to iPSCs and differentiating them to iHeps. Methods: Fresh human urine (250-500ml) was collected and the washed cell pellets were expanded in culture in a defined growth medium. The epithelial cells were reprogrammed

into iPSCs by using non-transgene integrating methods, such as delivering the pluripotency factor genes OCT3/4, SOX2, KLF4 and MYC by nucleofection of episomal (EBNA) plasmids or infection with recombinant Sendai viruses. After characterization of stable iPS cell lines, a 3-step differentiation toward hepatocytes was performed. At each step, the expression pattern of 141 genes was assessed by qRT PCR. Flow cytometry, immunocytochemistry and hepatocyte-specific functional assays were performed. Results: After 2 weeks of cultivation of urinary cells, 4-6 stable cell populations emerged. Both reprogramming strategies yielded iPSCs with characteristic features and normal karyotype. The first step selleck screening library of differentiation generated definitive endoderm cells, with 90% of the cells expressing the definitive endoderm marker Sox17, as shown by qRT PCR and immunocytochemistry. At the final stage, flow cytometry revealed that 86% and 29% of the cells were positive for human serum albumin and human asialoglycoprotein receptor, respectively.

The iHeps expressed mRNAs for nuclear receptors that regulate genes involved in cholesterol homeostasis, bile acid transport and detoxification, including farnesoid X receptor (FXR), and constitutive androstane receptor (CAR/ NR1l3), as well as the bile salt export pump (BSEP/ABCB11). The iHeps exhibited glycogen storage, and secreted urea and albumin Casein kinase 1 into the media. Conclusions: Urine cell-derived iPSCs can be reprogrammed and then efficiently differentiated to iHeps. Our methods allowed the expression of liver specific functions. Thus, urine is a readily available source for generating human hepatocyte-like cells that could be potentially useful for disease modeling, pharmacological development and regenerative medicine. Disclosures: The following people have nothing to disclose: Vanessa Sauer, Xia Wang, Krisztina Tar, Tatyana Tchaikowskaya, Yanfeng Li, Chandan Guha, Namita Roy-Chowdhury, Jayanta Roy-Chowdhury Microengineering human tissues on a chip remains an open challenge from both scientific and technological points of view.

“
“The mitochondrial cytochrome c oxidase I (cox1) gene has been promoted as a universal reference gene, or barcode, to identify organisms to the

species level. We evaluated whether cox1 would be appropriate to diagnose cetacean species. The 5′ end of cox1 (686 base pairs, bp) was sequenced for 46 of 86 recognized species of cetaceans. In addition, we included 105 sequences from GenBank, RAD001 clinical trial increasing our taxonomic coverage to 61 species. Particular focus was placed on sampling two subfamilies that contain closely related taxa: the Delphininae and the Globicephalinae. Species-specific sequences were observed for all but three taxa (Delphinus delphis, D. capensis, and Stenella coeruleoalba). Although correct assignment was seen for most species, significant overlap between intra- and interspecific variation makes cox1 an imperfect barcode for cetaceans. The efficacy of cox1 was compared to the 5′ end of the cytochrome b (cytb) gene, a mitochondrial region routinely used for cetacean species identification. Although cytb performed better than cox1 for some species, this marker could not differentiate other closely related taxa (Eubalaena spp.). Species identification for taxa not reliably identified check details using cox1 or cytb might be best addressed

through use of multiple mitochondrial DNA fragments or other newly developed markers. “
“Guidelines for sustainable tourism involving swimming with large whales are not well-developed compared to those focused on programs of swimming with delphinids. From September to November 2005 and August to September 2006, we collected behavioral and movement data

for southern right whales (Eubalaena australis) exposed to interactions with boats and swimmers at Península Valdés, Argentina. Whales were tracked from shore using a theodolite before, during, and after a series of directed interactions with swimmers and a boat. Resting, socializing, and surface active behavior decreased, traveling increased, and whales through swam faster and reoriented more often during interactions. Responses were variable by age/sex class, with mother/calf pairs showing strongest responses. Increased levels of tourism activity are a concern, as reduction in resting time and disruption of socialization among adults, juveniles, and mother/calf pairs have unknown long-term consequences. Additional data should be collected for whale behavior in proposed tourism and nontourism areas to build a long-term database which can be used to determine if reactions of whales change over time. Our data suggest that swimming with whales in Chubut Province should not be legalized until further investigations are completed, especially in light of the recent southern right whale die-offs recorded in Península Valdés.

[30] In the present study, ezetimibe not only decreased CCL4-induced

ROS production, but also increased protein expression of MTP check details and decreased ubiquitination of MTP and protein expression of Skp2. These in vitro findings suggest that the inhibition of ubiquitination and degradation of MTP protein via reduced hepatic protein levels of Skp2 and CDC20 observed in vivo was induced by a reduction of hepatic ROS by administration of ezetimibe. The limitation of our study is that the administration of ezetimibe was assessed only for 4 weeks, not for a longer or shorter period. Based on previous studies,[13, 14, 20] however, 4 weeks may be an optimal time to investigate the effect of ezetimibe to inhibit the development of NAFLD in FLS mice; thus, 4-week administration of ezetimibe was chosen in the current study. Further study will be considered to confirm the best period of ezetimibe administration. In conclusion, ezetimibe administration in a spontaneous model of NAFLD resulted in amelioration of histological lesions, hepatic ROS level and hepatic expression of lipogenesis-related genes. In addition, the FLS mice receiving ezetimibe showed reduced ubiquitinated MTP protein level together with lower protein levels of Skp2 and CDC20 in the liver. Ezetimibe treatment decreased intracellular ROS and ubiquitinated MTP protein

levels together with a lower protein level of Skp2, suggesting that ezetimibe suppressed post-translational degradation of MTP via a reduction of hepatic ROS generation in CCL4-induced MCA-RH7777 cells. These findings point to a beneficial effect of ezetimibe against NAFLD, possibly through DNA/RNA Synthesis inhibitor targeting hepatic ROS generation,

suggesting its potential Sclareol benefits in patients with NAFLD/NASH having low expression of MTP. WE GRATEFULLY THANK Ms T. Sato for her skillful technical assistance, and Dr T. Hirasawa (Shionogi & Company) for donating FLS mice. Figure S1 Food consumption and bodyweight variation. (a) Bodyweight variation of Fatty Liver Shionogi (FLS) mice fed normal diet. White squares, control group (CT). Black rhombuses, EZ. (b) Food consumption of FLS mice fed normal diet. White squares, CT. Black rhombuses, EZ. Data are expressed as mean ± standard deviation (n = 7). Figure S2 Effect of ezetimibe on glucose metabolism. A Glucose levels during ipGTT in Fatty Liver Shionogi (FLS) mice fed normal diet. White squares, control group. Black rhombuses, EZ. Table S1 The fatty acid content in liver of Fatty Liver Shionogi (FLS) mice fed a normal diet with or without ezetimibe. Data are expressed as mean ± SD; n = 4. CT = FLS mice fed a normal diet, EZ = FLS mice fed a normal diet containing ezetimibe. *P < 0.05 between CT and EZ. "
“Background and Study Aim:  Residual or locally recurrent lesions may occur after endoscopic therapy for epithelial colorectal tumors. Additional endoscopic mucosal resection is difficult for large lesions.

The loss of HDAC1 and/or HDAC2 (HDAC1/2) protein resulted in impaired liver regeneration. HDAC1/2 inactivation did not decrease hepatocytic 5-bromo-2-deoxyuridine uptake or the expression of proliferating cell nuclear antigen, cyclins, or cyclin-dependent

kinases. However, the levels of Ki67, a mitotic marker that is expressed from the mid-G1 phase to the end of mitosis and is closely involved in the regulation of mitotic progression, were greatly decreased, and abnormal mitosis lacking Ki67 expression was frequently observed in HDAC1/2-deficient livers. The down-regulation of either HDAC1/2 or Ki67 in the mouse liver cancer cell line Hepa1-6 resulted in similar mitotic defects. Finally, both HDAC1 and HDAC2 proteins were associated with the Ki67 gene mediated by CCAAT/enhancer-binding protein http://www.selleckchem.com/products/PLX-4032.html β. Conclusion: Both HDAC1 and HDAC2 play crucial roles in the regulation of liver regeneration. The loss of HDAC1/2 inhibits Ki67 expression

and Erlotinib price results in defective hepatocyte mitosis and impaired liver regeneration. (Hepatology 2013; 58:2089–2098) Histone deacetylases (HDACs) are a class of enzymes that remove acetyl groups from specific lysine residues on core histones and thereby regulate gene transcription through the structural modification of histones and chromatin.[1, 2] HDACs are recruited to multiprotein complexes on the genome and serve as epigenetic corepressors to facilitate the inhibition of target gene transcription; in this way, they regulate many physiological processes, including mitosis, apoptosis, and tumorigenesis.[3-5] The deregulation of HDACs is often associated with the development and progression of various cancers, and a number of HDAC inhibitors (HDACis) are currently being investigated for use in clinical tumor therapy.[6, 7] HDAC1 and HDAC2, the two members of the class I HDAC family, are ubiquitously expressed in organs and tissues, including the liver.[8]

Similar to other HDACs, neither bind directly to DNA; instead, Calpain HDAC1 and HDAC2 typically associate with corepressors, such as Sin3-SAP, NuRD, and CoREST, to form transcriptional corepressor complexes.[9] HDAC1 and/or HDAC2 (HDAC1/2) are also required for chromatin condensation, spindle formation, and chromosome separation during the mitotic phase of the cell cycle, and HDAC1/2 deregulation can lead to abnormal mitosis.[10-12] Most of the current knowledge regarding the role of HDAC1/2 has come from cancer research. A number of studies have used HDACis or small interfering RNA (siRNA) to investigate the role of HDAC1/2 in cell proliferation both in vivo and in vitro.

However, as noted above, the extent of variation in the supposedly dimorphic features was statistical (as opposed to presence/absence features of true dimorphism), and although they may have supported more conspicuous sexually dimorphic features in soft part anatomy that is not preserved, the statistical argument on the basis of hard parts is insufficient. The kind of variation appears much more akin to the sort of differences that characterize male and female crocodiles, which differ from each other mainly at

adult size, where it is www.selleckchem.com/ALK.html mostly a matter of relative robusticity (Webb et al., 1978; Chabreck & Joanen, 1979). If dimorphism were important in small basal ceratopsians, it should be emphasized or at least detectable in larger, more derived forms, but this does not seem to be the case. Lehman (1990) suggested a pattern of sexual dimorphism in Chasmosaurus and related species that could be traced through later ontogeny, but the small sample sizes, incomplete preservation, and lack

of association of much of this material, as Lehman noted, makes it difficult to evaluate hypotheses about sexual differences, even if they are accepted. Ryan et al.’s (2001) study of a ceratopsian bone bed, where dimorphism could be presumed to emerge, turned up no significant patterns. A recent review of Ceratopsia (Dodson et al., 2004) did not accept sexual dimorphism as a general feature in this clade of dinosaurs. Soft-part features and behaviors that are not preserved in extinct taxa may well have contributed to sexual selection (e.g. Sampson, 1997). selleck screening library However, to invoke them for extinct groups of dinosaurs is outside the pale of homological and analogical comparison. As for fossil birds, which are dinosaurs, we have almost no information about dimorphism; long tail feathers

in the basal avialian Confuciusornis are suggestive (Chiappe et al., 1999), but this is not enough to establish evolutionary polarity. Because dimorphism (and not just inter-sexual difference) is generally low in other reptiles (Fig. 5), the EPB does HSP90 not support sexual dimorphism in non-avian dinosaurs on the grounds of homological comparison. Vrba (1984) used the example of degree of horn differentiation, which is usually greater in alcelaphine bovids (hartebeest, wildebeest, etc.) than in the related aepycerotines (impalas), to suggest an explanation for the greater species diversity through time of the former clade. Sampson (1999) suggested that sexual selection, not just natural selection, could be the motor of enhanced diversity in certain subclades over others. He proposed a Mate Recognition Hypothesis (MRH) by which selection for positive recognition of mates could lead to increased differentiation of populations and eventually greater rates of speciation in some lineages over others.

A total of 523 hygromycin-resistant colonies were obtained, but some of the transformants appeared unstable and pSH75 might not have integrated into genomic DNA of host cells. To confirm stability, the transformants

were transferred five times to PDA containing 200 μg mL−1 hygromycin. Surviving transformants were subsequently grown on PDA without hygromycin for 3 days prior to being transferred to PDA with 200 μg mL−1 hygromycin. HDAC inhibitor In total, 323 transformants retained their resistance to hygromycin, and this indicated that they were stable. The colony morphology of these transformants changed as compared with the original strain of B. eleusines, and 98.4% of transformants were showed reduced growth for 1–3 days (Fig. 1). About 42.1% of colonies were grey to white compared with original black colonies of the fungus. Additionally, a small number of transformants did not sporulate (Table 1). Growth of transformant B014 was retarded after 1 day, colonies were grey and spore production was reduced to 50% of that of wild-type B. eleusines. Protoplasts of the wild-type B. eleusines were successfully transformed by linear plasmid DNA from the vector pSH75. When using circular plasmid DNA Staurosporine molecular weight without the restriction

enzyme, no transformant was obtained. The addition of XboI to the linear plasmid also showed a low transformation rate. However, addition of BamHI and HindIII to the linear plasmid significantly increased transformation efficiency, resulting in transformation rates of up to 4–5 transformants μg−1 plasmid (Table 2). Six toxin-deficient transformants were obtained. Mycelial growth of R. solani was effectively inhibited by the cell-free culture filtrate of wild-type B. eleusines, with Erlotinib mw a relative inhibitory rate of 89% (Table 3). However, the filtrate of the transformant B014 showed less inhibition and the colony diameter

of R. solani was close to that of the control after 24 h. This suggests that transformant B014 is deficient of the toxins. When sprayed with the filtrate of wild-type B. eleusines, barnyard grass was yellow 5 days after treatment (data not shown). However, when treated with the filtrate of transformant B014, no significant effect was observed in comparison with control. This result further demonstrated that B014 was no longer be able to produce phytotoxic metabolites against barnyard grass and therefore might be considered a toxin-deficient mutant of B. eleusines. Other transformants showed similar or only slightly reduced efficacy against barnyard grass relative to the wild-type. The metabolite chromatographic peaks in the wild-type sample (Fig. 2b) and five toxin-deficient mutants (data not shown) were close to the retention time (7.798 min) of the ophiobolin A standard (Fig. 2a, 7.778 min). The retention times were highly reproducible, varying by < 0.2 min. However, there was no detectable peak in the transformant B014 sample (Fig.