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Summary Heat stress induces misfolded protein accumulation in endoplasmic reticulum (ER), which initiates the unfolded protein response (UPR) in plants. Previous work has demonstrated the important role of a rice ER membrane‐associated transcription factor OsbZIP74 (also known as OsbZIP50) in UPR. However, how OsbZIP74 and other membrane‐associated transcription factors are involved in heat stress tolerance in rice is not reported. In the current study, we discovered that OsNTL3 is required for heat stress tolerance in rice. OsNTL3 is constitutively expressed and up‐regulated by heat and ER stresses. OsNTL3 encodes a NAC transcription factor with a predicted C‐terminal transmembrane domain. GFP‐OsNTL3 relocates from plasma membrane to nucleus in response to heat stress and ER stress inducers. Loss‐of‐function mutation of OsNTL3 confers heat sensitivity while inducible expression of the truncated form of OsNTL3 without the transmembrane domain increases heat tolerance in rice seedlings. RNA‐Seq analysis revealed that OsNTL3 regulates the expression of genes involved in ER protein folding and other processes. Interestingly, OsNTL3 directly binds to OsbZIP74 promoter and regulates its expression in response to heat stress. In turn, up‐regulation of OsNTL3 by heat stress is dependent on OsbZIP74. Thus, our work reveals the important role of OsNTL3 in thermotolerance, and a regulatory circuit mediated by OsbZIP74 and OsNTL3 in communications among ER, plasma membrane and nucleus under heat stress conditions.

Chloroplasts are important for plant growth and development. RNA editing in chloroplast converts cytidines (Cs) to uridines (Us) at specific transcript positions and provides a correction mechanism to restore conserved codons or creates start or stop codons. However, the underlined molecular mechanism is not yet fully understood. In the present study, we identified a thermo-sensitive mutant in leaf color 1 (tsl1) and found that TSL1 is allelic to DELAYED GREENING 1 (DG1). The missense mutation of DG1 in tsl1 mutant confers a high temperature sensitivity and impaired chloroplast development at an elevated ambient temperature in Arabidopsis. Subsequent analysis showed that chloroplast RNA editing at several sites including accD-1568, ndhD-2, and petL-5 is impaired in tsl1 mutant plants grown at an elevated temperature. DG1 interacts with MORF2 and other proteins such as DYW1 and DYW2 involved in chloroplast RNA editing. In vitro RNA electrophoretic mobility shift assay demonstrated that DG1 binds to RNA targets such as accD, ndhD, and petL. Thus, our results revealed that DG1 is important for maintaining chloroplast mRNA editing in Arabidopsis.

This paper reveals that XBAT31 ubiquitinates and degrades ELF3 to attenuate its inhibitory effects on PIF4., Elevated ambient temperature has wide effects on plant growth and development. ELF3, a proposed thermosensor, negatively regulates protein activity of the growth-promoting factor PIF4, and such an inhibitory effect is subjected to attenuation at warm temperature. However, how ELF3 stability is regulated at warm temperature remains enigmatic. Here, we report the identification of XBAT31 as the E3 ligase that mediates ELF3 degradation in response to warm temperature in Arabidopsis. XBAT31 interacts with ELF3, ubiquitinates ELF3, and promotes ELF3 degradation via the 26S proteasome. Mutation of XBAT31 results in enhanced accumulation of ELF3 and reduced hypocotyl elongation at warm temperature. In contrast, overexpression of XBAT31 accelerates ELF3 degradation and promotes hypocotyl growth. Furthermore, XBAT31 interacts with the B-box protein BBX18, and the XBAT31-mediated ELF3 degradation is dependent on BBX18. Thus, our findings reveal that XBAT31-mediated destruction of ELF3 represents an additional regulatory layer of complexity in temperature signaling during plant thermomorphogenesis.

Plants are capable of coordination of their growth and development with ambient temperatures. EARLY FLOWERING3 (ELF3), an essential component of plant circadian clock, is also involved in ambient temperature sensing, as well as in inhibiting the expression and protein activity of the thermoresponsive regulator Phytochrome interacting factor 4 (PIF4). The ELF3 activity is subjected to attenuation in response to warm temperature, however, how the protein level of ELF3 is regulated at warm temperature remains less understood. Here, we report that the E3 ligase XB3 ORTHOLOG 5 IN ARABIDOPSIS THALIANA, XBAT35, mediates ELF3 degradation. XBAT35 interacts with ELF3 and ubiquitinates ELF3. Loss-of-function mutation of XBAT35 increases the protein level of ELF3 and confers a short-hypocotyl phenotype under warm temperature conditions. Thus, our findings establish that XBAT35 mediates ELF3 degradation to lift the inhibition of ELF3 on PIF4 for promoting thermoresponsive hypocotyl growth in plants. This article is protected by copyright. All rights reserved.

Background Colorectal cancer (CRC) is a common malignant gastrointestinal tumor. In China, CRC is the 5th most commonly diagnosed cancer. The vast majority of CRC cases are sporadic and evolve with the adenoma-carcinoma sequence. There is mounting evidence indicating that gut microbiota and inflammation play important roles in the development of CRC although study results are not entirely consistent. In the current study, we investigated the changes in the CRC-associated bacteria and plasma inflammatory factors and their relationships based on data from a case-control study of Han Chinese. We included 130 initially diagnosed CRC patients, 88 advanced colorectal adenoma patients (A-CRA), 62 patients with benign intestinal polyps and 130 controls. Results Fecal microbiota composition was obtained using 16S ribosomal DNA (16S rDNA) sequencing. PCOA analysis showed structural differences in microbiota among the four study groups (P = 0.001, Unweighted Unifrac). Twenty-four CRC-associated bacteria were selected by a two-step statistical method and significant correlations were observed within these microbes. CRC-associated bacteria were found to change with the degree of malignancy. Plasma C-reactive protein (CRP) and soluble tumor necrosis factor II (sTNFR-II) displayed significant differences among the four study groups and increased with adenoma-carcinoma sequence. The correlations of CRP and sTNFR-II with several CRC-associated microbes were also explored. Conclusions CRC-associated species and plasma inflammatory factors tended to change along the adenoma-carcinoma sequence. Several CRC-associated bacteria were correlated with CRP and sTNFR-II. It is likely that gut microbiome and inflammation gradually form a microenvironment that is associated with CRC development. Electronic supplementary material The online version of this article (10.1186/s12866-018-1232-6) contains supplementary material, which is available to authorized users.

Background/Aims: The aim of this study was to investigate the influence of Cx43- and Smad-mediated TGF-β/BMP signaling pathway on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into cartilage and inhibition of ossification. Methods: BMSCs of Wistar rats were cultured and assigned into 5 groups for transfection with adenoviruses. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were employed to detect mRNA and protein expressions of target genes. The condition of cartilage and ossification were measured by a series of staining methods. Subcutaneous injection of mesenchymal stem cells (MSCs) into nude rats was performed. Results: After transfection, compared to the AdGFP group, the corresponding target mRNAs were overexpressed in the AdBMP2, AdSmad1, AdCx43 + AdSmad1 and AdCx43 + AdSmad1 + AdBMP2 groups, and overexpression of BMP2 at the mRNA and protein expression was observed in the AdSmad1 and AdCx43 + AdSmad1 groups. The mRNA expressions of aggrecan (ACAN) and collagen type II alpha 1 (Col2a1), the glycosaminoglycan content of the extracellular matrix and the expression of type II collagen, Col2a1, osteopontin (OPN) and osteocalcin (OC) were higher in the AdBMP2, AdSmad1, AdCx43 + AdSmad1 and AdCx43 + AdSmad1 + AdBMP2 groups than in the AdGFP group; alkaline phosphatase (ALP) activity and mRNA and protein expressions of Runx2 were also higher in these groups than in the AdGFP group. Heterotopic osteogenesis tests demonstrated evident cartilage differentiation ability in the AdCx43 + AdSmad1 + AdBMP2 groups. In comparison, the AdCx43 + AdSmad1 and AdSmad1 groups exhibited weaker cartilage differentiation abilities. Conclusion: Cx43 and Smad1 promote BMP-induced cartilage differentiation of BMSCs and inhibit osteoblast differentiation, which provide a new strategy for cartilage tissue engineering using exogenous Cx43 and Smad1.

Background Gut microbiota is closely related to age. Studies from Europe and the U.S. identified featured microbiota in different age groups for the elderly. Asian studies mainly focused on people living in longevity areas. Featured microbiota for the elderly people of different age groups, especially in the centenarian in the general population, has not been well investigated in China. Method We conducted a comparative study by including 198 subjects of three age groups (65–70, 90–99, and 100+ years) in East China. Information regarding age, sex, height, weight, waist circumference, hip circumference, food preference, smoking status and alcohol consumption were collected by using a structured questionnaire. Fecal samples for each participant were collected as well. 16S rRNA gene sequencing were employed to analyze the gut microbiota composition. Logistic regression with LASSO feature selection was used to identify featured taxa in different age groups and to assess their potential interactions with other factors such as lifestyle. Result The gut microbiota of the 90–99 year and 100+ year age groups showed more diversity, robustness, and richness compared with the 65–70 year age group. PCoA analysis showed a clear separation between the 65–70 and 100+ year age groups. At the species level, Bacteroides fragilis, Parabacteroides merdae, Ruminococcus gnavus, Coprococcus and Clostridium perfringens increased, but Bacteroides vulgatus, Ruminococcus sp.5139BFAA and Clostridium sp.AT5 decreased in the 90–99 year age group. The age differences in gut microbiota were similar across the strata of smoking, alcohol consumption status and food preference. Conclusion Our study demonstrated age differences in many aspects of gut microbiota, such as overall diversity, microbiota structure, and relative abundance of key taxa. Moreover, the gut microbiota of centenarian was significantly different from those of younger age groups of the elderly.

Many sources of stress cause accumulation of unfolded or misfolded proteins in endoplasmic reticulum (ER), which elicits the unfolded protein response (UPR) to either promote cell survival or programmed cell death depending on different developmental context or stress severity. The Arabidopsis membrane-associated transcription factor, bZIP28, is the functional equivalent of mammalian ATF6, which relocates from the ER to the Golgi where it is proteolytically processed and released from the membrane to the nucleus to mediate the UPR. Although the canonical site-1 protease (S1P) cleavage site on the ER lumen-facing domain is well conserved between bZIP28 and ATF6, the importance of S1P cleavage on bZIP28 has not been experimentally demonstrated. Here we provide genetic evidence that the RRIL(573) site, but not the RVLM(373) site, on the lumen-facing domain of bZIP28 is critical for the biological function of bZIP28 under ER stress condition. Further biochemistry and cell biology studies demonstrated that the RRIL(573) site, but not the RVLM(373) site, is required for proteolytic processing and nuclear relocation of bZIP28 in response to ER stress. Our results reveal that S1P cleavage site plays a pivotal role in activation and function of bZIP28 during UPR in plants.

The present study investigates cytogenetic damage in lymphocytes, derived from three victims who were unfortunately exposed to cobalt-60 (60Co) radiation (the 1999 accident occurred in a village in China’s Henan province). Case A of the three victims was exposed to a higher dose of 60Co radiation than Cases B and C. The chromosomal aberrations, cytokinesis-block micronucleus (CBMN, the CBMN assay), and DNA double-strand breaks (DSBs, the comet assay) examined in this study are biomarkers for cytogenetic abnormalities. After the lymphocytes collected from the victims were cultured, the frequencies of dicentric chromosomes and rings (dic + r) and CBMN in the first mitotic division detected in the lymphocytes of Case A were found to be substantially higher than in Cases B and C. Similarly, the DNA-DSB level found in the peripheral blood collected from Case A was much higher than those of Cases B and C. These results suggest that an acutely enhanced induction of the 60Co-induced cytogenetic abnormality frequency in humans depends on the dose of 60Co radiation. This finding is supported by the data obtained using practical techniques to evaluate early lymphoid-tissue abnormalities induced after exposure to acute radiation.

Toll-like receptors (TLRs) are involved in the progression of ischemic brain injury and hence vascular dementia; however, the underlying mechanisms are largely unknown. Here, we have investigated the interrelationship between stress-responsive heme oxygenase (HO)-1 isoenzyme and TLR4 during chronic brain hypoperfusion. The right unilateral common carotid artery occlusion was performed by ligation of the right common carotid artery in C57BL/6J mice. The brain cortex or hippocampus was removed for western blotting and confocal immunofluorescence analysis. The link between HO-1 and TLR4 was further examined by silencing TLR4 and pharmacological inhibition of HO-1 in primary cultured cortical neurons. Cognitive dysfunction and decrease in cerebral blood flow in mice were observed 4 weeks after the occlusion. Our data further show that common carotid artery occlusion induced an increase in TLR4 and HO-1 protein levels. Although the administration of CoPP (10 mg/kg), HO-1 agonist, improved the cognitive dysfunction in a mice model of occlusion, western blot analysis in primary cultured cortical neurons showed that HO-1 was upregulated after lipopolysaccharide treatment; this was partially abolished by the TLR4 siRNA interference. The flow cytometry analysis showed that pharmacological inhibition of HO-1 by ZnPP (100 μM) further exaggerated lipopolysaccharide-induced neuronal cell death. Hence, stress-responsive HO-1 isoenzyme participates in TLR4-induced inflammation during chronic brain ischemia. The pharmacological manipulation of TLR4 or the HO-1 antioxidant defense pathway may represent a novel treatment strategy for neuronal protection in vascular dementia.

Arsenic, the 20th most abundant element in the earth crust, is one of the oldest drugs in the world. It was used in the 18th century in treating hematopoietic malignancies, discarded in 1950s in favor of chemotherapeutic agents (busulphan and others), and was revived in the 1970s due to its dramatic efficacy on acute promyelocytic leukemia (APL) driven by the t(15;17) translocation-generated PML-RARα fusion. Arsenic represents the most potent single agent for APL, and achieves a five-year overall survival of 90% in APL patients when combined with all-trans retinoic acid (ATRA) and chemotherapy (daunorubicin and cytarabine), turning this disease from highly fatal to highly curable. Arsenic triggers sumoylation/ubiquitination and proteasomal degradation of PML-RARα via directly binding to the C3HC4 zinc finger motif in the RBCC domain of the PML moiety and induction of its homodimerization/multimerization and interaction with the SUMO E2 conjugase Ubc9. Because of its multiplicity of targets and complex mechanisms of action, arsenic is widely tested in combination with other agents in a variety of malignancies. Other arsenic containing recipes including oral formulations and organic arsenicals are being developed and tested, and progress in these areas will definitely expand the use of arsenicals in other malignant diseases.

Arsenic, a curative agent for acute promyelocytic leukemia, induces cell apoptosis and degradation of BCR-ABL in chronic myelogenous leukemia (CML). We demonstrated that ubiquitination and degradation of BCR-ABL was mediated by c-CBL, a RING-type E3 ligase that was also shown to be involved in ubiquitination for many other receptor/protein tyrosine kinases. Our data showed that c-CBL protein was considerably up-regulated by arsenic sulfide (As 4 S 4 ). Interestingly, arsenic directly bound the RING finger domain of c-CBL to inhibit its self-ubiquitination/degradation without interfering with the enhancement of ubiquitination and subsequent proteolysis of its substrate BCR-ABL. Degradation of BCR-ABL due to c-CBL induction as a result of arsenic treatment was also observed in vivo in CML mice. These findings provide insight into the molecular mechanisms of arsenic and further support its therapeutic applications in CML in combination with tyrosine kinase inhibitors and potentially also in other malignancies involving aberrant receptor/protein tyrosine kinase signaling.

The association between primary biliary cirrhosis (PBC) and autoimmune hemolytic anemia (AIHA) is uncommon; only fourteen such case reports have been described. In this report, three patients who developed AIHA on the basis of PBC underwent successful therapy with corticosteroids and ursodeoxycholic acid (UDCA). Patient 3 was more complicated, suffering from PBC, Evans syndrome, Sjögren syndrome and Klinefelter syndrome simultaneously. This has not previously been reported in the world literature. Review of all fifteen cases showed that there is a prominent occurrence sequence that AIHA might take place on the basis of PBC. With sufficient doses of corticosteroids or immunosuppressant therapy, besides hemolysis under effective control, liver function also improved. According to the criteria of secondary AIHA, we may call them PBC-related AIHA. Thus, patients with PBC with serum bilirubin levels rising suddenly should undergo screening for associated hemolysis. Recommended treatment for PBC-related AIHA includes sufficient doses of corticosteroids to control the hemolysis in the acute phase, and immunosuppressant or adequate dose of UDCA to maintain therapy. These case reports have been increasing in recent years, so further reserch is needed to illustrate the incidence and natural courses of these two organ-specific autoimmune diseases.

Membrane-associated basic-leucine zipper (bZIP) transcription factors that reside in the endoplasmic reticulum represent a newly described class of plant stress sensor/transducers. The bZIP factors are anchored in endoplasmic reticulum (ER) membranes with their C-terminal tails facing the ER lumen and their N terminii, which contain transcriptional components, facing the cytosol. In response to stress, cytosolic components of the transcription factors are released by proteolysis and move to the nucleus where they promote the up-regulation of stress response genes. One such stress sensor/transducer in Arabidopsis is AtbZIP17, which is activated in response to salt stress. With the aim of enhancing salt tolerance, a constitutively activated form of AtbZIP17, truncated before its membrane-anchor domain, was introduced into transgenic plants. When placed under the control of a constitutive promoter, the activated form of AtbZIP17 up-regulated stress response genes under unstressed conditions, but caused a substantial delay in plant development. When the activated form of AtbZIP17 was placed under the control of stress-inducible promoter, development was normal under unstressed conditions. Under salt stress conditions, the stress-inducible expression of the activated AtbZIP17 enhanced salt tolerance as demonstrated by chlorophyll bleaching and seedling survival assays.

Phytosulfokines (PSKs) are secreted, sulfated peptide hormones derived from larger prepropeptide precursors. Proteolytic processing of one of the precursors, AtPSK4, was demonstrated by cleavage of a preproAtPSK4-myc transgene product to AtPSK4-myc. Cleavage of proAtPSK4 was induced by placing root explants in tissue culture. The processing of proAtPSK4 was dependent on AtSBT1.1, a subtilisin-like serine protease, encoded by one of 56 subtilase genes in Arabidopsis. The gene encoding AtSBT1.1 was up-regulated following the transfer of root explants to tissue culture, suggesting that activation of the proteolytic machinery that cleaves proAtPSK4 is dependent on AtSBT1.1 expression. We also demonstrated that a fluorogenic peptide representing the putative subtilase recognition site in proAtPSK4 is cleaved in vitro by affinity-purified AtSBT1.1. An alanine scan through the recognition site peptide indicated that AtSBT1.1 is fairly specific for the AtPSK4 precursor. Thus, this peptide growth factor, which promotes callus formation in culture, is proteolytically cleaved from its precursor by a specific plant subtilase encoded by a gene that is up-regulated during the process of transferring root explants to tissue culture.

Ag+-doped TiO2 films on stainless steel were prepared by a sol-gel method and their microstructures and compositions were studied with X-Ray Diffractometer, Scanning Electron Microscope (SEM) and X-ray Photoelectron Spectroscopy. It was shown that Fe atoms in untreated stainless steel react with Ag+ in the TiO2 film and form FeTiO3, which has an acicular crystal form under SEM observation. As a result, Ag+ in the film is reduced to the silver atom, which degrades the antibacterial property of the film. However, after an oxidization of the substrate, a layer of ferric oxide is formed, which reacts with Fe atoms that would otherwise react with and reduce Ag+, and then forms FeTiO3. Thus, the penetration of Fe atoms is stopped and Ag+ in the anatase-structure TiO2 film is protected from the reduction, which enhanced antibacterial property of the film.

Senile dementia (SD) is a syndrome characterized by progressive neurological deterioration. Treatment for the disease is still under investigation. Bamboo leaf extract (B-extract) has been known for its biological efficacy in anti-oxidant and anti-cancer activities. However, study on B-extract for its protection against dementia is very limited. The effect of B-extract on a rat model with SD was examined. B-extract improved spatial learning ability of the dementia rats. The hippocampus of dementia model rats showed reduced levels of acetylcholine (ACh), epinephrine (E), norepinephrine (NE), and dopamine (DA), and increased activities of acetylcholine esterase (AChE) and monoamine oxidase (MAO). Treatment with B-extract 20 mg/(kg·d) for 7 weeks significantly inhibited the enzyme activity compared with untreated dementia rats, and raised the levels of ACh, E, and DA in the hippocampus. In addition, treatment with B-extract elevated the level of γ-aminobutyric acid (GABA), but reduced the level of glutamate (Glu) in the brain. These data suggest that B-extract might be a potential drug in treating impairment of spatial memory in dementia rats by regulating the central neurotransmitter function.

Field-based screens for genetic variation in reproductive-stage drought tolerance are often confounded by genetic variation in root depth, flowering date and biomass at flowering. To reduce these confounding effects and to impose drought stress more precisely, we grew contrasting genotypes of rice (Oryza sativaL.) in shallow containers offlooded soil. When water was withheld from the shallow-rooted indica genotype IR64 for 6 days starting at � 17, � 7, 0, 10 and 20 days after heading (DAH), the impact on grainyield was greatest at � 7 and 0DAH. The most sensitive yield component was filled grain percentage (spikelet fertility). Data on yield and spikelet fertility were also obtained for another shallow-rooted lowland indica (BRRI Dhan 31) and two deep-rooted upland tropical japonicas (Azucena and Moroberekan). Compared with well-watered controls, withholding of water for 6 days reduced spikelet fertility by � 80% in IR64, BRRI Dhan 31 and Azucena but by 22% in Moroberekan. In a detailed comparison, water was withheld from IR64 for 5 days and from Moroberekan for 6 days, starting 3 days before the heading date of well-watered controls. Under these stress conditions, heading was delayed for 4 days in IR64 and 6 days in Moroberekan. Spikelet fertility in the top four rachis branches was reduced by 80% in IR64 and 16% in Moroberekan, a difference attributable principally to reduced anther dehiscence and lower stigma pollen density in IR64. Two properties of Moroberekan may contribute to high anther dehiscence after stress/re-watering: (i) constitutively superior development of fibrous structures in the endothecium at the anther apex and base and (ii) better maintenance of pollen size. All ovaries of Moroberekan received pollen—an average of 60 grains in wellwatered plants and 42 grains in stressed/re-watered plants. In well-watered IR64, all ovaries received an average of 31 grains, whereas in stressed/re-watered IR64, 67% of ovaries received no pollen and the remainder received an average of eight grains. Moroberekan may, therefore, be a source of reproductive-stage drought tolerance through genes that maintain anther dehiscence during recovery from low water status.

Certain antibiotics detected in urine are associated with childhood obesity. In the current experimental study, we investigated two representative antibiotics detected in urine, florfenicol and azithromycin, for their early effects on adipogenesis, gut microbiota, short-chain fatty acids (SCFAs), and bile acids in mice. Thirty C57BL/6 mice aged four weeks were randomly divided into three groups (florfenicol, azithromycin and control). The two experimental groups were administered florfenicol or azithromycin at 5 mg/kg/day for four weeks. Body weight was measured weekly. The composition of the gut microbiota, body fat, SCFAs, and bile acids in colon contents were measured at the end of the experiment. The composition of the gut microbiota was determined by sequencing the bacterial 16S rRNA gene. The concentration of SCFAs and bile acids was determined using gas chromatography and liquid chromatography coupled to tandem mass spectrometry, respectively. The composition of the gut microbiota indicated that the two antibiotics altered the gut microbiota composition and decreased its richness and diversity. At the phylum level, the ratio of Firmicutes/Bacteroidetes increased significantly in the antibiotic groups. At the genus level, there were declines in Christensenella, Gordonibacter and Anaerotruncus in the florfenicol group, in Lactobacillus in the azithromycin group, and in Alistipes, Desulfovibrio, Parasutterella and Rikenella in both the antibiotic groups. The decrease in Rikenella in the azithromycin group was particularly noticeable. The concentration of SCFAs and secondary bile acids decreased in the colon, but the concentration of primary bile acids increased. These findings indicated that florfenicol and azithromycin increased adipogenesis and altered gut microbiota composition, SCFA production, and bile acid metabolism, suggesting that exposure to antibiotics might be one risk factor for childhood obesity. More studies are needed to investigate the specific mechanisms.

Background: It has been reported that zinc sulphate contributes an anti-inflammatory action in many animal models; however, the impact of zinc in colitis remains unclear. The aim of the present study was to examine the role of zinc sulphate in experimental colitis. Methods: Colitis was induced by 2,4,6-trinitrobenzenesulphonic acid (TNB) in rats. Beginning at the first day of TNB colitis, the rats were treated with a zinc sulphate enema once daily for 6 days. The rats were examined 8 days later. Results: The TNB induced severe colitis as evidenced by increased mucosal lesion area, mucosal myeloperoxidase (MPO) activity and prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) levels. Six days after the application of the zinc sulphate enema, the mucosal lesion area, MPO activity, PGE2 and LTB4 levels all decreased significantly. Mucosal superoxide dismutase activity remained unchanged after zinc treatments. Conclusions: Our data suggest that zinc sulphate enemas have an anti-inflammatory action on experimental colitis. © 1999 Blackwell Science Asia Pty Ltd

Thymocyte apoptosis was assessed by counting apoptotic bodies with flow cytometry (FCM) and measuring DNA fragmentation with fluorescence spectrophotometry (FSP). J-shaped dose-response curves were obtained after both whole-body irradiation (WBI) of mice and in vitro irradiation of EL4 cells with doses ranging from 0.025 to 4 Gy X-rays. There was a significant reduction of apoptosis rate to below control level with doses within 0.2 Gy, and a dose-dependent increase in apoptosis with doses above 0.5 Gy. When thymocytes were cultured 24 h after WBI with 75 mGy X-rays in complete RPMI 1640 medium, a reduction in apoptosis was observed in the course of incubation for 72 h, and the presence of Con A in the medium accentuated this reduction in a dose- and time-dependent manner. The implications of these observations and the possible molecular mechanisms for future studies are proposed.

Prenatal exposure to chlorpyrifos (CPF) leads to cognitive impairments in adulthood. The cytoarchitectural basis is unclear. In the present study, we assessed the effects of prenatal CPF exposure on T-maze delayed alternation task and the win-shift/lose-shift responses associated with the morphology of the dorsal hippocampus (dHPC) and the medial prefrontal cortex (mPFC) in adult animals. Gestational ICR female mice were exposed to 0, 1 or 5 mg/kg/d of CPF through gestational days 13–17. Behavioral experiments were performed on postnatal days (PD) 45–60 of the male and female offsprings; morphological samples were collected on PD 60. Our behavioral study results showed a gradual increase in the number of lose-shift errors on increased memory loads in the 5 mg/kg/d CPF-treated males. A weak initial increase in the number of lose-shift errors was observed in the females. In all of the groups, no significant differences were observed in the number of win-shift errors and correct of the first choice. The morphological studies showed extensive condensed nucleus and enlarged intercellular spaces in the CA1 and DG sub-regions in the dHPC of the CPF-treated males and the DG sub-region of the CPF-treated females. The cell count was significantly reduced in these sub-regions. The morphological studies showed no obvious abnormalities at PrL and IL of mPFC in the CPF-treated males and females, but the cell count was reduced. Our findings suggest that prenatal CPF exposure at 5 mg/kg/d induces selective cognitive impairments, which based on the morphological deficits in the dHPC and the mPFC.

Nearly 60% of acute myeloid leukemia (AML) patients with the t(8;21)(q22;q22) translocation fail to achieve long-term disease-free survival. Our previous studies demonstrated that oridonin selectively induces apoptosis of t(8;21) leukemia cells and causes cleavage of AML1-ETO oncoprotein resulting from t(8;21), but the underlying mechanisms remain unclear. We show that oridonin interacted with glutathione and thioredoxin/thioredoxin reductase to increase intracellular reactive oxygen species, which in turn activated caspase-3 in t(8;21) cells. Moreover, oridonin bound AML1-ETO, directing the enzymatic cleavage at aspartic acid 188 via caspase-3 to generate a truncated AML1-ETO (ΔAML1-ETO) and preventing the protein from further proteolysis. ΔAML1-ETO interacted with AML1-ETO and interfered with the trans-regulatory functions of remaining AML1-ETO oncoprotein, thus acting as a tumor suppressor that mediates the anti-leukemia effect of oridonin. Furthermore, oridonin inhibited the activity of c-Kit(+) leukemia-initiating cells. Therefore, oridonin is a potential lead compound for molecular target-based therapy of leukemia.

Crop yield is most sensitive to water deficit during the reproductive stage. For rice, the most sensitive yield component is spikelet fertility and the most sensitive stage is immediately before heading. Here, we examined the effect of drought on the anther proteome of two rice genotypes: Moroberekan and IR64. Water was withheld for 3 d before heading (3DBH) in well watered controls for 5 d until the flag leaf relative water content (RWC) had declined to 45–50%. Plants were then re-watered and heading occurred 2–3 d later, representing a delay of 4–5 d relative to controls. The anther proteins were separated at 3 DBH, at the end of the stress period, and at heading in stressed/re-watered plants and controls by two-dimensional (2-D) gel electrophoresis, and 93 protein spots were affected reproducibly in abundance by drought during the experiment across two rice genotypes. After drought stress, upon re-watering, expressions of 24 protein spots were irreversible in both genotypes, 60 protein spots were irreversible in IR64 but reversible in Moroberekan, only nine protein spots were irreversible in Moroberekan while reversible in IR64. Among them, there were 14 newly drought-induced protein spots in IR64; none of them was reversible on re-watering. However, there were 13 newly drought-induced protein spots in Moroberekan, 10 of them were reversible on re-watering, including six drought-induced protein spots that were not reversed in IR64. Taken together, our proteomics data reveal that drought-tolerant genotype Moroberekan possessed better recovery capability following drought and re-watering at the anther proteome level than the drought-sensitive genotype IR64. The disruptions of drought to rice anther development and pollen cell functions are also discussed in the paper.

There is an evidence that the Kato-Katz technique lacks sensitivity and may hence be an unsuitable method for the assessment of the 'real infection status' in community with low-intensity infections. In this study, six Kato-Katz thick smears (examination of two stool samples with three thick smears each) were used as the diagnostic 'gold' standard for estimating the prevalence of Schistosoma japonicum infection and the results were compared with results based on fewer Kato-Katz thick smear readings. A total of 1055 individuals in 2005 and 725 in 2006 from an endemic village were recruited for the study. The observed prevalence increased gradually with the number of Kato-Katz thick smears examined, and hence the rate of underestimation decreased accordingly. The prevalence based on single Kato-Katz thick smear readings was significantly lower than that obtained using five or six thick smears. The rate of underestimation based on using two and three Kato-Katz thick smears, a typical diagnostic effort in the national schistosomiasis control programme, was about 36.0% (28.4-48.9%) and 25.0% (15.9-40.7%). The number of Kato-Katz thick smears required to secure detection of a S. japonicum infection varies with the infection intensity level. Indeed, examination of a single thick smear was sufficient when the geometric mean of the fecal content of eggs per gram (EPG) was 250 or higher in infected individuals, while six Kato-Katz thick smears were required when the EPG score was lower than 10. In conclusion, our results confirm that the prevalence of S. japonicum infection in a community is generally considerably "underestimated". Moreover, our findings provide a benchmark for the proper application of the Kato-Katz technique and the rational evaluation of the epidemic situation, as well as a scientific basis for constructing a mathematic diagnostic model.

To investigate the effects of electroacupuncture (EA) at neiguan (PC6) on gastric distention-induced transient lower esophageal sphincter relaxations (TLESRs) and discuss the mechanisms of this treatment.Protocol I: Twelve healthy cats underwent gastric distention for 60 min on the first day. Electrical acupoint stimulation was applied at the neiguan or a sham point on the hip in randomized order before gastric distention, on the third day and fifth day. Those cats that underwent EA at neiguan on the fifth day were named "Neiguan Group" and the cats that underwent EA at a sham acupoint on the fifth day were named "Sham Group" (control group). During the experiment the frequency of TLESRs and lower esophageal sphincter (LES) pressure were observed by a perfused sleeve assembly. Plasma levels of gastrin (GAS) and motilin (MTL) were determined by radioimmunoassay. Nitrite/nitrate concentration in plasma and tissues were measured by Griess reagent. The nuclei in the brain stem were observed by immunohistochemistry method of c-Fos and NADPH-d dyeing. Protocol II: Thirty six healthy cats were divided into 6 groups randomly. We gave saline (2 mL iv. control group), phaclofen (5 mg/kg iv. GABA-B antagonist), cholecystokinin octapeptide (CCK-8) (1 microg/kg per hour iv.), L-Arginine (200 mg/kg iv.), naloxone (2.5 micromol/kg iv.) and tacrine (5.6 mg/kg ip. cholinesterase inhibitor) respectively before EA at Neiguan and gastric distention. And the frequencies of TLESRs in experimental groups were compared with the control group.Protocol I: Not only the frequency of gastric distention-induced TLESR in 60 min but also the rate of common cavity during TLESRs were significantly decreased by EA at neiguan compared to that of sham acupoint stimulation. C-Fos immunoreactivity and NOS reactivity in the solitarius (NTS) and dorsal motor nucleus of the vagus (DMV) were significantly decreased by EA at neiguan compared to that of the sham group. However, the positive nuclei of C-Fos and NOS in reticular formation of the medulla (RFM) were increased by EA at neiguan. Protocol II: The inhibited effect of EA at neiguan on TLESR's frequency was completely restored by pretreatment with CCK (23.5/h vs 4.5/h, P0.05), L-arginine (17.5/h vs 4.5/h, P0.05) and naloxone(12/h vs 4.5/h, P0.05). On the contrary, phaclofen (6/h vs 4.5/h, P0.05) and tacrine (9.5/h vs 4.5/h, P0.05) did not influence it.Electric acupoint stimulation at Neiguan significantly inhibits the frequency of TLESR and the rate of common cavity during TLESR in cats. This effect appears to act on the brain stem, and may be mediated through nitric oxide (NO), CCK-A receptor and mu-opioid receptors. But the GABAB receptor and acetylcholine may not be involved in it.

SUMMARY. The present study explores the changes of nitric oxide synthesis and esophageal dysmotility in a feline model of esophagitis. Perfusion of the esophagus with acid produced inflammatory changes of esophageal mucosa. The esophageal motility was measured before and after the perfusion. The nitric oxide synthase activity, the l-arginine uptake, and the content of cyclic guanine monophosphate of the muscle and the mucous membrane were determined and the NADPH-diaphorase was stained. Esophagitis impairs the motility of the esophagus. The nitric oxide synthase activity, the content of cyclic guanine monophosphate, the NADPH-diaphorase stain and the maximum velocity of l-arginine uptake of lower esophageal sphincter of the cats in the acid perfusion group were higher than those of the control group. The maximum velocity of l-arginine transport and the content of cyclic guanine monophosphate of the mucosa in the acid perfusion group were lower than those of the control group. The results suggested that during esophagitis there is an alteration of the l-arginine/nitric oxide synthase/nitric oxide pathway in the esophagus, which may be one of the important mechanisms of esophageal motility dysfunction.

Fifty-eight acute promyelocytic leukemia (APL) patients (11 newly diagnosed and 47 relapsed) were studied for arsenic trioxide (As2O3) treatment. Clinical complete remission (CR) was obtained in 8 of 11 (72.7%) newly diagnosed cases. However, As2O3 treatment resulted in hepatic toxicity in 7 cases including 2 deaths, in contrast to the mild liver dysfunction in one third of the relapsed patients. Forty of forty-seven (85.1%) relapsed patients achieved CR. Two of three nonresponders showed clonal evolution at relapse, with disappearance of t(15;17) and PML-RAR fusion gene in 1 and shift to a dominant AML-1-ETO population in another, suggesting a correlation between PML-RAR expression and therapeutic response. In a follow-up of 33 relapsed cases over 7 to 48 months, the estimated disease-free survival (DFS) rates for 1 and 2 years were 63.6% and 41.6%, respectively, and the actual median DFS was 17 months. Patients with white blood cell (WBC) count below 10 × 109/L at relapse had better survival than those with WBC count over 10 × 109/L (P = .038). The duration of As2O3-induced CR was related to postremission therapy, because there was only 2 of 11 relapses in patients treated with As2O3 combined with chemotherapy, compared with 12 of 18 relapses with As2O3 alone (P = .01). Reverse transcription polymerase chain reaction (RT-PCR) analysis in both newly diagnosed and relapsed groups showed long-term use of As2O3 could lead to a molecular remission in some patients. We thus recommend that ATRA be used as first choice for remission induction in newly diagnosed APL cases, whereas As2O3 can be either used as a rescue for relapsed cases or included into multidrug consolidation/maintenance clinical trials.

Idiopathic voiding disorders affect up to 10–15% of men and women. We describe bladder abnormalities in mice with targeted deletion of the gene for neuronal nitric oxide synthase which model the clinical disorders. The mice possess hypertrophic dilated bladders and dysfunctional urinary outlets which do not relax in response to electrical field stimulation or L-arginine. The mice also display increased urinary frequency.

Abstract 1200 Using immunoprecipitation (IP)-2D-nano-HPLC-MALDI-MS-MS, we identified c-CBL in association with BCR-ABL in a multi-protein complex in K562 cells. In vitro ubiquitination and mutagenesis analyses show that c-CBL serves as a specific E3 ligase for ubiquitination of BCR-ABL at K1517. Arsenic sulfide (As4S4) treatment results in increased c-CBL protein level, which promotes ubiquitination and subsequent degradation of BCR-ABL and apoptosis of K562 cells. Elevated c-CBL is necessary and sufficient to recapitulate the effect of As4S4. Interestingly, arsenic directly binds the RING finger domain of c-CBL, inhibiting its self-ubiquitination and degradation, thus leading to accumulation of c-CBL. However, this interaction between As4S4 and c-CBL does not interfere with its E3 ligase activity towards BCR-ABL. Increased c-CBL protein and BCR-ABL degradation are also observed in vivo after As4S4 administration in BCR-ABL leukemia mice. These findings provide insights into the molecular mechanisms of arsenic and its potential therapeutic applications in CML. Disclosures: No relevant conflicts of interest to declare.

AIM: To investigate the effect of pinaverium bromide, a L-type calcium channel blocker with selectivity for the gastrointestinal tract on contractile activity of colonic circular smooth muscle in normal or cold-restraint stressed rats and its possible mechanism. METHODS: Cold-restraint stress was conducted on rats to increase fecal pellets output. Each isolated colonic circular muscle strip was suspended in a tissue chamber containing warm oxygenated Tyrode-Ringer solution. The contractile response to ACh or KCl was measured isometrically on ink-writing recorder. Incubated muscle in different concentrations of pinaverium and the effects of pinaverium were investigated on ACh or KCl-induced contraction. Colon smooth muscle cells were cultured from rats and [Ca2+]i was measured in cell suspension using the Ca2+ fluorescent dye fura-2/AM. RESULTS: During stress, rats fecal pellet output increased 61% (P < 0.01). Stimulated with ACh or KCl, the muscle contractility was higher in stress than that in control. Pinaverium inhibited the increment of [Ca2+]i and the muscle contraction in response to ACh or KCl in a dose dependent manner. A significant inhibition of pinaverium to ACh or KCl induced [Ca2+]i increment was observed at 10-6 mol/L. The IC50 values for inhibition of ACh induced contraction for the stress and control group were 1.66 × 10-6 mol/L and 0.91 × 10-6 mol/L, respectively. The IC50 values for inhibition of KCl induced contraction for the stress and control group were 8.13 × 10-7 mol/L and 3.80 × 10-7 mol/L, respectively. CONCLUSION: Increase in [Ca2+]i of smooth muscle cells is directly related to the generation of contraction force in colon. L-type Ca2+ channels represent the main route of Ca2+ entry. Pinaverium inhibits the calcium influx through L-type channels; decreases the contractile response to many kinds of agonists and regulates the stress-induced colon hypermotility.