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Zheng Wang,1 Zi-Ming Gao,2 Hai-Bo Huang,2 Li-Sha Sun,2 An-Qi Sun,2 Kai Li2 1Department of Otorhinolaryngology, the First Affiliated Hospital of China Medical University, Shenyang 110001, China; 2 Department of Surgical Oncology, the First Affiliated Hospital of China Medical University, Shenyang 110001, China Purpose: No consensus exists on the impact of polymorphisms in cytokines (such as interleukin IL-8 and IL-18) on cancer risk; moreover, there is very little evidence regarding head and neck cancer (HNC). Methods: Thus, a meta-analysis including 22 studies with 4731 cases and 8736 controls was conducted to evaluate this association. The summary odds ratio (OR) and corresponding 95% confidence intervals (CIs) for C-X-C motif chemokine ligand 8 (CXCL8, which encodes IL-8) and IL-18 polymorphisms and HNC risk were estimated. Results: The results showed a significantly increased risk of HNC susceptibility for IL18-137 G/C in five genetic models, but, interestingly, no significant association was found for the CXCL8-251 A/T polymorphism. When stratified by cancer type, an increased risk of nasopharyngeal cancer was found for both -137 G/C and -251A/T. When the studies were stratified by ethnicity and genotyping method, there were significant associations between Asian populations and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) studies for -137 G/C, and African populations for -251 A/T in some genetic models. A positive association was also found between the population-based groups in some models for -137 G/C; conversely, significantly decreased risk was found among the -251 A/T hospital-based group. Meta-regression was also conducted. The publication year, control source, and cancer type contributed to CXCL8 -251 A/T heterogeneity; however, no factors were found that contributed to IL-18 -137 G/C heterogeneity. Marginal significance was found in the recessive model for IL-18 -137 G/C by Egger’s test, whereas no publication bias was detected for CXCL8 -251 A/T. Conclusions: The results indicate that the IL-18 -137 G/C polymorphism is associated with HNC risk, especially nasopharyngeal cancer, in Asian populations and, when using PCR-RFLP, CXCL8 -251 A/T polymorphisms play a complex role in HNC development. Keywords: interleukin-8, interleukin-18, polymorphism, head and neck cancer, meta-analysis

Background: Dietary fiber (DF) intake may have a protective effect against type 2 diabetes (T2D); however, its relationship with diabetic kidney disease (DKD) remains unclear., Aim: To investigate the potential association between DF intake and the prevalence of DKD in individuals diagnosed with T2D., Methods: This cross-sectional study used data from the National Health and Nutrition Examination Survey collected between 2005 and 2018. DF intake was assessed through 24-h dietary recall interviews, and DKD diagnosis in individuals with T2D was based on predefined criteria, including albuminuria, impaired glomerular filtration rate, or a combination of both. Logistic regression analysis was used to assess the association between DF intake and DKD, and comprehensive subgroup and sensitivity analyses were performed., Results: Among the 6032 participants, 38.4% had DKD. With lower DF intake-T1 (≤ 6.4 g/1000 kcal/day)-as a reference, the adjusted odds ratio for DF and DKD for levels T2 (6.5-10.0 g/1000 kcal/day) and T3 (≥ 10.1 g/1000 kcal/day) were 0.97 (95%CI: 0.84-1.12, P = 0.674) and 0.79 (95%CI: 0.68-0.92, P = 0.002), respectively. The subgroup analysis yielded consistent results across various demographic and health-related subgroups, with no statistically significant interactions (all P > 0.05)., Conclusion: In United States adults with T2D, increased DF intake may be related to reduced DKD incidence. Further research is required to confirm these findings., Competing Interests: Conflict-of-interest statement: There are no conflicts of interest to report., (©The Author(s) 2024. Published by Baishideng Publishing Group Inc. All rights reserved.)

Lactic acid bacteria (LAB) are recognized as food-grade safe microorganisms and have many beneficial effects. LAB could maintain the host intestinal homeostasis and regulate intestinal microbial community to exert antibacterial effects. In this study, Lactiplantibacillus plantarum (L. plantarum, Lp01) strain isolated from pig intestine was orally administered to C57BL/6 mice, and mice were then infected with Salmonella typhimurium (ATCC14028). The protective effects of L. plantarum were evaluated by monitoring body weight loss, survival rates, bacterial loads in tissue, colon histopathology analysis, and cytokine secretion. 16S rRNA gene sequencing was also utilized to detect the dynamics of the blind gut microbial community in mice. We found that L. plantarum could significantly reduce the body weight loss and improve the survival rates. The survival rate in the L. P-Sty group was up to 67.5%, which was much higher than that in the STY group (25%). Counting of bacterial loads displayed that the colony-forming unit (CFU) of S. typhimurium in the spleen (p < 0.05) and the liver (p < 0.05) from L. P-Sty group both decreased, compared with STY group. Intestinal histopathology showed that it alleviated the intestinal injury caused by Salmonella, inhibited the secretion of pro-inflammatory cytokines, and promoted anti-inflammatory cytokines (p < 0. 01). In addition, L. plantarum also significantly ameliorated the intestinal gut microbiome disturbance caused by Salmonella. It displayed an obvious increase of beneficial bacteria including Lactobacillus and Bacteroidetes and reduction of pathogenic bacteria like Proteobacteria. In conclusion, L. plantarum could regulate microbial community to inhibit Salmonella typhimurium infection., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Rice paddy soil is a hotspot of antibiotic resistance genes (ARGs) due to the application of organic fertilizers. However, the temporal dynamics of ARGs in rice paddy soil and its flooded water during the growing season remain underexplored. In this study, a microcosm experiment was conducted to explore the ARG profiles in a long term (130 days) flooded two-phase manure-amended soil-water system. By using high-throughput quantitative PCR array, a total of 23-98 and 34-85 ARGs were detected in the soil and overlying water, respectively. Regression analysis exhibited significant negative correlations between ARG profile similarities and flooding duration, indicating that flooding significantly altered the resistome (P < 0.001). This finding was validated by the increased ARG abundance in the soil and the overlying water, for example, after 130 days flooding, the abundance of ARGs in CK soil was increased from 0.03 to 1.20 copies per 16S rRNA. The PCoA analysis further suggested pig manure application resulted in distinct ARG profiles in the soil-water continuum compared with those of the non-amended control (Adonis, P < 0.05). The Venn diagram showed that all ARGs detected in the pig manure were present in the treated soil. Twelve ARGs (e.g., sul1) were shared among the pig manure, manure-amended soil, and overlying water, indicating that certain manure- or soil-borne ARGs were readily dispersed from the soil to the overlying water. Moreover, the enhanced relationships between the ARGs and mobile genetic elements in pig manure applied soil-water continuum indicate that the application of organic matter could accelerate the emergence and dissemination of ARGs. These findings suggested that flooding represents a crucial pathway for dispersal of ARGs from the soil to the overlying water. Identification of highly mobile ARGs in the soil-water continuum is essential for assessing their potential risk to human health and promoting the development of sustainable agricultural practices to mitigate their spread., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Despite the importance of soil bacterial and fungal communities for ecosystem services and human welfare, how their ecological networks respond to climatic aridity have yet been evaluated. Here, we collected soil samples from 47 sites across 2500 km in coastal and inland areas of eastern Australia with contrasting status of aridity. We found that the diversity of both bacteria and fungi significantly differed between inland and coastal soils. Despite the significant differences in soil nutrient availability and stoichiometry between the inland and coastal regions, aridity was the most important predictor of bacterial and fungal community compositions. Aridity has altered the potential microbial migration rates and further impacted the microbial assembly processes by increasing the importance of stochasticity in bacterial and fungal communities. More importantly, ecological network analysis indicated that aridity enhanced the complexity and stability of the bacterial network but reduced that of the fungal network, possibly due to the contrasting impacts of aridity on the community-level habitat niche breadth and overlaps. Our work paves the way towards a more comprehensive understanding of how climate changes will alter soil microbial communities, which is integral to predicting their long-term consequences for ecosystem sustainability and resilience to future disturbances., (© 2022 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Soil protists are key in regulating soil microbial communities. However, our understanding on the role of soil protists in shaping antibiotic resistome is limited. Here, we considered the diversity and composition of bacteria, fungi and protists in arable soils collected from a long-term field experiment with multiple fertilization treatments. We explored the effects of soil protists on antibiotic resistome using high-throughput qPCR. Our results showed that long term fertilization had stronger effect on the composition of protists than those of bacteria and fungi. The detected number and relative abundance of antibiotic resistance genes (ARGs) were elevated in soils amended with organic fertilizer. Co-occurrence network analysis revealed that changes in protists may contribute to the changes in ARGs composition, and the application of different fertilizers altered the communities of protistan consumers, suggesting that effects of protistan communities on ARGs might be altered by the top-down impact on bacterial composition. This study demonstrates soil protists as promising agents in monitoring and regulating ecological risk of antibiotic resistome associated with organic fertilizers., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Conjunctive transfer of antibiotic resistance genes (ARGs) among bacteria driven by plasmids facilitated the evolution and spread of antibiotic resistance. Heavy metal exposure accelerated the plasmid-mediated conjunctive transfer of ARGs. Nanomaterials are well-known adsorbents for heavy metals removal, with the capability of combatting resistant bacteria/facilitating conjunctive transfer of ARGs. However, co-effect of heavy metals and nanomaterials on plasmid-mediated conjunctive transfer of ARGs was still unknown. In this study, we investigated the effect of the simultaneous exposure of Cd 2+ and nano Fe 2 O 3 on conjugative transfer of plasmid RP4 from Pseudomonas putida KT2442 to water microbial community. The permeability of bacterial cell membranes, antioxidant enzyme activities and conjugation gene expression were also investigated. The results suggested that the combination of Cd 2+ and high concentration nano Fe 2 O 3 (10 mg/L and 100 mg/L) significantly increased conjugative transfer frequencies of RP4 plasmid (p < 0.05). The most transconjugants were detected in the treatment of co-exposure to Cd 2+ and nano Fe 2 O 3 , the majority of which were identified to be human pathogens. The mechanisms of the exacerbated conjugative transfer of ARGs were involved in the enhancement of cell membrane permeability, antioxidant enzyme activities, and mRNA expression levels of the conjugation genes by the co-effect of Cd 2+ and nano Fe 2 O 3 . This study confirmed that the simultaneous exposure to Cd 2+ and nano Fe 2 O 3 exerted a synergetic co-effect on plasmid-mediated conjunctive transfer of ARGs, emphasizing that the co-effect of nanomaterials and heavy metals should be prudently evaluated when combating antibiotic resistance., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Pulmonary vessel intimal sarcoma (IS) is rare., Methods: We studied gross pathology, microscopic images and immunohistochemistry of 2 pulmonary artery ISs (PAISs) and 2 pulmonary vein ISs (PVISs), followed up the prognosis. The clinical manifestations of IS, imaging examination, electrocardiographic examination and serological examination were also studied., Results: Grossly, the tumors were grayish yellow or grayish-white accompanied by local bleeding, and were manifested as sticky and slippery nodules. PAISs were located in the lumen of the pulmonary trunk or right ventricular outflow tract, and 1 of them had pedicle. PVISs were mainly located in the left atrium. Microscopically, the heteromorphic spindle cells were arranged in lobules and bundles, partially epithelioid, with visible mitotic figures. There were interstitial mucoid degeneration and local hemorrhage and necrosis/infarction. Immunohistochemistry showed that vimentin, h-caldesmon and MDM2 were all positive, SATB2 (+, 3/4); Ki67 proliferation rate was 30-60%; smooth muscle actin, desmin and CD56 were partially positive; cytokeratin and CD34 were locally positive; CD31, FLI-1, and ERG vascular markers were negative; and S-100 was negative. Case 4 showed MDM2 (12q15) amplification. Tumor markers were negative in venous blood; and lactate dehydrogenase increased in 2 cases. 3 patients died after surgery, 1 still survives after 14 months with lung and chest metastases for immunotherapy and 9 courses of chemotherapy., Conclusions: IS is rare. Microscopically, it is mainly composed of spindle cells (or local epithelioid), along with interstitial mucoid degeneration. IS can differentiate into tumor of fibroblasts, bone, cartilage and smooth muscle, etc. The prognosis is poor., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://dx.doi.org/10.21037/tcr-20-3468). The authors have no conflicts of interest to declare., (2021 Translational Cancer Research. All rights reserved.)

Background: Fatty liver disease (FLD) is associated with increased risk for hepatocellular carcinoma (HCC) and is associated with rising rates of diabetes and obesity. The prevalence of FLD is rising among Asian American and Pacific Islanders (AAPIs) and Latinos. This study examined health literacy, knowledge, and risk factors for FLD among AAPIs and Latinos in Los Angeles., Methods: Data from in-person interviews and clinical measures (body mass index (BMI), body fat percentage, and blood pressure) were obtained from adults aged 18-82 years at four health fairs from November 2018 to March 2019. Interviews assessed knowledge about FLD, access to health resources, and satisfaction with current physician. Correct responses to knowledge questions were summed to generate a FLD knowledge score. Linear regression models were used to examine the association between knowledge score and age, sex, and race/ethnicity., Results: A total of 102 subjects were AAPI and 33 were Latino. Over 65% of participants had heard of FLD but demonstrated limited knowledge about FLD. Only 24% of subjects reported receiving FLD resources in their preferred language. Most subjects failed to identify several risk factors and key symptoms of FLD. Mean knowledge score for subjects who had heard of FLD was 7.58 (95% CI 7.15-8.01) out of a possible 16 points, and for those who had not who had not heard of FLD it was 5.71 (5.00-6.42) (p <0.0001)., Conclusions: A lack of culturally competent resources and effective communication strategies between physicians and patients regarding FLD contributes to a lower awareness about the increased risk of FLD among AAPIs and Latinos. Future studies should investigate optimal methods to educate these communities about FLD and its associations with HCC.

Rhizosphere microbiota play a pivotal role in promoting plant growth and defending against pathogens, but their responses to abiotic environmental stress remain largely elusive. Here, we investigated the influences of low-N stress on rhizosphere bacteria of six sorghum cultivars in a glasshouse experiment. The alpha diversity of bacteria (as revealed by Shannon diversity and Chao1 richness indices) was remarkably lower in rhizosphere soils than in bulk soils, and was significantly higher under low-N stress than under N addition. Principal coordinates analysis revealed that the bacterial community compositions in rhizosphere soils were clearly separated from bulk soils, and the rhizosphere soils under low-N stress or with N fertilization were clearly separated, indicating that both rhizosphere effects and N fertilization impacted the rhizosphere bacterial community. Notably, the relative abundances of beneficial bacteria such as Bacillaceae and Streptomycetaceae significantly increased in rhizosphere soils under low-N stress, which had significantly positive correlations with the sorghum N uptake. The relative abundance of Nitrosomonadaceae in rhizosphere soils was significantly lower than that in bulk soils, while the relative abundance of Rhizobiaceae showed an opposite pattern. Taken together, our results suggested that sorghum rhizosphere effects can reduce soil bacterial diversity possibly through recruiting specific bacterial species under low N stress., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Salidroside is a phenolic secondary metabolite present in plants of the genus Rhodiola, and studies investigating its extensive pharmacological activities and mechanisms have recently attracted increasing attention. This review summarizes the progress of recent research on the antiproliferative activities of salidroside and its effects on breast, ovarian, cervical, colorectal, lung, liver, gastric, bladder, renal, and skin cancer as well as gliomas and fibrosarcomas. Thus, it provides a reference for the further development and utilization of salidroside.

Background: Although Borrmann type IV (B-4) gastric cancer has a higher mortality rate and presents distant metastasis easily, especially peritoneal metastasis, when diagnosed, some B-4 patients were found to have no distant metastasis by preoperative detection and underwent curative surgery, which was defined as circumscribed B-4 in our study. In this study, we focused on the circumscribed B-4 patients without distant metastasis during surgery to identify factors related to prognosis and postoperative peritoneal cavity metastasis (PPCM), which is important for selecting an appropriate therapeutic strategy., Aim: To identify factors related to the prognosis and PPCM of B-4 patients., Methods: A total of 117 B-4 patients who underwent gastrectomy between January 2005 and December 2012 were included in this study. Survival analysis was performed using Kaplan-Meier analysis and Cox multivariate models. Pearson correlation analyses were performed to identify the factors related to PPCM. All statistical analyses were performed using SPSS 20.0., Results: Lymph node status, gastrectomy type, and postoperative chemotherapy were independent prognostic factors in 117 circumscribed B-4 patients. Subtotal gastrectomy combined with chemotherapy could significantly improve the long-term survival time. Six patients who were diagnosed with pN0 and received the combination therapy had a 3-year survival rate of 100% and a median survival of 77.7 mo. Even for patients with metastatic lymph nodes ( n = 13), the combination therapy also increased the 3-year overall survival rate to 57.1%. In addition, positive lymph node status was the only factor ( P = 0.005) correlated with PPCM in certain B-4 patients, and chemotherapy was useful for suppressing PPCM in patients with subtotal gastrectomy but not in those with total gastrectomy., Conclusion: Lymph node status is an independent prognostic factor for circumscribed B-4 patients. In addition, subtotal gastrectomy and postoperative chemotherapy could effectively improve prognosis and even suppress PPCM., Competing Interests: Conflict-of-interest statement: All authors declare no conflicts of interest., (©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.)

Disturbed blood flow has been recognized to promote platelet aggregation and thrombosis via increasing accumulation of von Willebrand factor (VWF) at the arterial post-stenotic sites. The mechanism underlying the disturbed-flow regulated endothelial VWF production remains elusive. Here we described a mouse model, in which the left external carotid artery (LECA) is ligated to generate disturbed flow in the common carotid artery. Ligation of LECA increased VWF accumulation in the plasma. Carotid arterial thrombosis was induced by ferric chloride (FeCl 3 ) application and the time to occlusion in the ligated vessels was reduced in comparison with the unligated vessels. In vitro , endothelial cells were subjected to oscillatory shear (OS, 0.5 ± 4 dynes/cm 2 ) or pulsatile shear (PS, 12 ± 4 dynes/cm 2 ). OS promoted VWF secretion as well as the cell conditioned media-induced platelet aggregation by regulating the intracellular localization of vesicle-associated membrane protein 3 (VAMP3) and synaptosomal-associated protein 23 (SNAP23). Disruption of vimentin intermediate filaments abolished the OS-induced translocation of SNAP23 to the cell membrane. Knockdown of VAMP3 and SNAP23 reduced the endothelial secretion of VWF. Systemic inhibition of VAMP3 and SNAP23 by treatment of mice with rapamycin significantly ameliorated the FeCl 3 -induced thrombogenesis, whereas intraluminal overexpression of VAMP3 and SNAP23 aggravated it. Our findings demonstrate VAMP3 and SNAP23 as potential targets for preventing the disturbed flow-accelerated thrombus formation., (Copyright © 2020 Zhu, Jiang, Liu, Xi, Wang, Yang, Yao, Pang, Han, Zhang, Sun and Zhou.)

Objective: We investigated the antitumor effects of salidroside and preliminarily examined its underlying mechanisms by establishing a nude mouse model bearing MCF-7 breast cancer cell xenografts., Methods: The mice were grouped and intraperitoneally injected with salidroside, paclitaxel, or physiological saline. Tumor samples were weighed, and immunohistochemical staining with hematoxylin and eosin and anti-CD34 antibody was performed. Tumor cell apoptosis was observed using the terminal deoxynucleotidyl transferase deoxyuridine dUTP nick end labeling assay. Bcl-1, p53, Bax, and caspase 3 expression in tumor tissues was determined via western blotting., Results: The tumor inhibition rate of high-dose salidroside was 75.16%, which was significantly higher than the rates for paclitaxel and saline. A tumor tissue pathology analysis revealed that high-dose salidroside inhibited tumor cell proliferation and promoted tumor cell apoptosis. Western blotting revealed that Bcl-2 and p53 expression were significantly lower in the salidroside group than in the other groups, whereas Bax and caspase 3 (17 kDa) expression were increased., Conclusions: Salidroside was more effective than paclitaxel in inhibiting tumor growth in MCF-7 breast cancer cell-bearing nude mice. The mechanism of action may involve Bcl-2 and p53 downregulation and Bax and caspase 3 upregulation, thereby increasing proapoptotic factor expression and inducing tumor cell apoptosis.

Background: Radical gastrectomy with D2 lymph node (LN) dissection is the standard surgical procedure for patients with resectable gastric cancer (GC). In the fifteenth edition of the Japanese Classification of Gastric Carcinoma, the 14v LN (LNs along the root of the superior mesenteric vein) was defined as the regional gastric LN. The efficacy of 14v LN dissection during radical distal gastrectomy for lower-third GC remains controversial., Aim: To analyze whether the addition of 14v LN dissection improved the survival of patients with lower-third GC., Methods: The data from 65 patients who underwent 14v LN dissection and 65 patients treated without 14v LN dissection were selected using the propensity score-matched method from our institute database constructed between 2000 and 2012. Overall survival was compared between the groups., Results: Overall survival was similar between patients with 14v LN metastasis and those with distant metastasis ( P = 0.521). Among patients with pathological stage IIIA disease, those who were treated with 14v LN dissection had a significantly higher overall survival than those treated without it ( P = 0.020). Multivariate analysis showed that age < 65 years and pT2-3 stage were independent favorable prognostic factors for prolonged overall survival in patients with pathological stage IIIA disease. Patients with No. 1, No. 6, No. 8a, or No. 11p LN metastasis were at higher risk of having 14v LN metastasis., Conclusion: Adding 14v LN dissection to D2 dissection during radical distal gastrectomy may improve the overall survival of patients with pathological stage IIIA lower-third GC., Competing Interests: Conflict-of-interest statement: All authors declare no conflicts of interest., (©The Author(s) 2019. Published by Baishideng Publishing Group Inc. All rights reserved.)

A diverse array of ARGs has been detected in the guts of soil fauna residing in farmland soil. Biochar has been widely used in farmland for soil remediation and improvement of soil quality; however, the effects of biochar amendment on the gut-associated ARGs of soil fauna remain unclear. In the present study, collembolans were cultivated in soils amended with 6 types of biochars. High-throughput qPCR was used to establish ARG profiles of the collembolan guts as well as the surrounding soils. A total of 73 and 162 subtypes of ARGs were detected in the collembolan guts and soils, respectively. Biochar amendment significantly altered the ARG compositions of the collembolan guts and soils, in a biochar quality-dependent manner. However, only manure-derived biochar, which contained elevated concentrations of heavy metals, increased the relative abundance of gut-associated ARGs. Changes in the gut microbial community, MGEs and biochar properties explained 84% of the total ARG variations in the collembolan guts. The findings of this study suggested that biochar properties should receive more attention, as high doses of heavy metals in biochar could increase the abundance of ARGs in collembolan guts, thereby contributing to the spread of ARGs in the environment through collembolan movement., (Copyright © 2019 Elsevier B.V. All rights reserved.)

( S)-2,15-Br 2 -DHTP-catalyzed asymmetric conjugate addition of boronic acids to β-trifluoromethyl α,β-unsaturated ketones and enones was studied. The reaction afforded the corresponding Michael addition products in moderate to high yields with excellent enantioselectivities (up to 99:1 er). This catalytic system features mild reaction conditions, high efficiency, and tolerance to heteroarylboronic acids.

Diets of soil-feeding earthworms contain abundant nitrate and iron(III) oxides, which are potential electron acceptors for mineralization of organic compounds. The earthworm gut provides an ideal habitat for ingested iron(III)-reducing microorganisms. However, little is known about iron(III) reduction and its interaction with other processes in the guts of earthworms. Here, we determined the dynamics of iron(III) and revealed its interaction with the turnover of organic acids and nitrate in the gut of the earthworm Pheretima guillelmi. Samples from gut contents combined with anoxic incubation were used for chemical analysis and 16S rRNA based Illumina sequencing. Chemical analysis showed that higher ratios of iron(II)/iron(III), nitrite/nitrate, and more abundant organic acids were contained in the in vivo gut of the earthworm P. guillelmi than those in the in situ soil. A higher rate of iron(III) reduction was detected in treatments of microcosmic incubation with gut contents (IG gut) than that with soil (IG soil), and nitrate reduction occurred earlier than iron(III) reduction in both treatments. Potential iron(III) reducers were dominated by fermentative genera Clostridium, Bacillus, and Desulfotomaculum in the treatment of IG gut, while they were dominated by dissimilatory iron(III)-reducing genera Geobacter in the treatment of IG soil. The iron(III)-reducing microbial community shared several genera with denitrifers in the treatment of IG gut, revealing a close link between iron(III) reduction and denitrification in the gut of earthworms. Collectively, our findings demonstrated that iron(III) reduction occurred along the gut and provided novel insights into the great contribution of earthworm gut microbiota on Fe and the associated C and N cycling in soil environments.

Two wheat cultivars (Jimai 22 and Shannong 23) were selected to investigate the changes of seed vigor at different development stages in different years by standard germination test, and the effect of environmental temperature on it was also analyzed, which would provide a theoretical basis for the utilization of early wheat seeds and the production of high vigor seeds. The results showed that the germination ability of fresh seeds appeared around 26 d after anthesis, and then the germination rate of fresh seeds displayed an overall upward trend with the development of wheat seeds. The germination energy, germination rate and germination index of dry seeds rapidly increased from 5 d to 8 d after anthesis, and then remained relatively stable, while the vigor index increased continually and reached the peak 4-6 days before the end of ripening due to the increase of seedling dry mass. The dry seeds at different developmental stages were also studied by field-plan-ting and the seed vigor of their progenies was also determined. For Jimai 22, the results indicated that the field emergence of dry seeds at 17 d after anthesis was higher and the plant could form grains, there were no significant differences in germination rate and vigor index among different samples of progeny seeds. In addition, the changes of seed vigor at different development stages were significantly affected by the environmental temperature. In the year with the high mean daily average temperature, high mean daily maximum temperature, high mean daily minimum temperature after anthesis, and large mean daily temperature difference after anthesis, the seed development time was shorter, but the 100-grain mass and seed vigor reached the peak earlier; Otherwise, the seed development time was longer, but the accumulated temperature of ripe stage was higher, and the seed vigor was higher.

This study aimed to analyze the enzyme activities and gene expression differences related to seed germination in different wheat genotypes, and to clarify the relationship between seed vigor and the related enzyme activities as well as gene expression under stress germination conditions. We measured seed vigor, total soluble sugar content, soluble protein content, α-amylase activity, cysteine protease activity and gene expression of the related enzymes of four wheat cultivars under drought, artificial aging and cold soaking stress. Results showed that drought, artificial aging and cold soaking stress affected seed vigor to some extent. Under different germination conditions, total soluble sugar content showed an increasing trend with small amplitude at first and then decreased with small amplitude and after that increased rapidly again, while soluble protein content in the four cultivars gradually decreased with germination time. The α-amylase activity of the four cultivars showed a rising trend on the whole, but that of Yunong 949 and Lunxuan 061 declined after germinating for 60 hours after cold soaking stress. Cysteine protease activity decreased at first and then increased as a whole. However, under drought stress condition, cysteine protease activity of Yunong 949, Yumai 49-198 and Lunxuan 061 increased at first and then decreased and finally increased again. The α-AMY (α-amylase gene) expression levels increased at first and then decreased as a whole under different germination conditions. The expression level of α-AMY in Lunxuan 061 after cold soaking stress was higher than that of CK, while the α-AMY expression levels in four cultivars under other stress germination conditions were lower than that of CK. The expression level of CP (cysteine protease gene) showed an increasing trend. No significant difference of CP expression level was found in Chang 4738 between artificial aging treatment and CK. The CP expression levels in the four cultivars under other stress conditions were higher than that of CK. The results demonstrated that there was no direct relationship between the enzyme activities and gene expression levels of α-amylase and cysteine protease under different germination conditions. The α-amylase activity and total soluble sugar content had an extremely significant positive correlation, but the correlation between cysteine protease activity and soluble protein content was not significant. The α-amylase activity significantly positively correlated with vigor index under standard germination condition. However, the α-amylase activity had no significant correlation with vigor index under stress conditions. After cold soaking stress, cysteine protease activity significantly positively correlated with vi-gor index during seed germination, but the correlation was not significant under standard germination, drought stress and artificial aging.

A total of 16 wheat cultivars were selected to detect seed vigor of different genotypes using standard germination test, seed germination test under stress conditions and field emergence test. The adversity resistance indices of seed vigor indices and field emergence percentage under different germination conditions were used as the indices to evaluate adversity resistance. Principal component analysis and cluster analysis were used for the comprehensive evaluation of seed vigor. Results showed that drought stress, artificial aging and cold soaking treatments affected seed vigor to some extent. The adversity resistance indices of the artificial aging and cold soaking tests were significantly positively correlated with the field emergence percentage, while the adversity resistance index of drought stress test had no significant correlation with the field emergence percentage. 16 wheat cultivars were classified as three groups based on the principal component analysis and cluster analysis. Yunong 949, Yumai 49-198, Luyuan 502, Zhengyumai 9987, Shimai 21, Shannong 23, and Shixin 828 belonged to high vigor seeds. Xunong 5, Yunong 982, Tangmai 8, Jimai 20, Jimai 22, Jinan 17, and Shannong 20 belonged to medium vigor seeds. The other two cultivars, Chang 4738 and Lunxuan 061, belonged to low vigor seeds.

The heteromeric membrane protein Organic Solute Transporter alpha/beta is the major bile acid efflux transporter in the intestine. Physical association of its alpha and beta subunits is essential for their polarized basolateral membrane localization and function in the transport of bile acids and other organic solutes. We identified a highly conserved acidic dileucine motif (-EL20L21EE) at the extracellular amino-tail of organic solute transporter beta from multiple species. To characterize the role of this protein interacting domain in the association of the human beta and alpha subunits and in membrane localization of the transporter, Leu20 and Leu21 on the amino-tail of human organic solute transporter beta were replaced with alanines by site-directed mutagenesis. Co-immunoprecipitation study in HEK293 cells demonstrated that substitution of the leucine residues with alanines prevented the interaction of the human beta mutant with the alpha subunit. Membrane biotinylation demonstrated that the LL/AA mutant eliminated membrane expression of both subunits. Computational-based modelling of human organic solute transporter beta suggested that the LL/AA mutation substantially alters both the structure and lipophilicity of the surface, thereby not only affecting the interaction with the alpha subunit but also possibly impacting the capacity of the beta subunit to traffick through the cell and interact with the membrane. In summary, our findings indicate that the dileucine motif in the extracellular N-terminal region of human organic solute transporter beta subunit plays a critical role in the association with the alpha subunit and in its polarized plasma membrane localization.

The transgenic pollen spread is the main pathway of transgenic plant gene flow. The maximum distance of pollen dispersal (horizontal), the spatial dynamics of pollen movement (vertical), and the patterns of pollen dispersal are important considerations in biosafety assessments of genetically modified crops. To evaluate wheat (Triticum aestivum) pollen dispersal, we measured the pollen suspension velocity and analyzed pollen dispersal patterns under natural conditions in the Huanghuai River wheat-growing region in 2009. The pollen suspension velocity was 0.3-0.4 m/s. The highest pollen densities were detected in the north, northwest, and south of the pollen source. Pollen was dispersed over distances greater than 245 m in the northwest and northeast directions. At the pollen source center, pollen density decreased with increasing vertical height. In the north of the pollen source, the pollen density from 65 m to 225 m showed a wave-mode decrease with increasing height. The horizontal transport of pollen over longer distances fitted polynomial equations. In the north, the pollen density was highest at 45 m from the pollen source, and decreased with increasing distance. In the northwest, the pollen density showed a double-peak trend. In the northeast, pollen density was highest from 45 m to 125 m from the source. Wind speeds greater than the pollen suspension velocity and the duration of continuous gusts were the main factors affecting pollen dispersal. This information will be useful for determining the spatial isolation distances for hybrid seed production and for the commercial production of transgenic wheat.

The organic solute transporter-α/β (OSTα/β) is a heteromeric transporter that is essential for bile acid and sterol disposition and for the enterohepatic circulation. To better understand the mechanism underlying OST gene regulation, the effects of retinoic acid (RA) on OSTα/β gene expression were investigated. The results show a dose-dependent induction of OSTβ but not OSTα expression in both Huh7 and HepG2 cells by RA treatment. A novel functional RA receptor response element (RARE; so-called DR5) in the promoter of OSTβ gene was identified. The interaction of RARα/RXRα with the RARE was verified by electrophoretic mobility shift and chromatin immunoprecipitation assays and its functional importance by hOSTβ promoter activation in luciferase reporter assays. The studies demonstrated that the RARE is also a constitutive androstane receptor (CAR) binding site for OSTβ gene regulation. These results suggest that OSTβ is a target of both FXR-mediated (by binding to IR-1 element) and RARα- and CAR-mediated (by binding to DR5 element) gene regulation pathways. In summary, this study has uncovered a novel RARE (DR5) element in the promoter of OSTβ that binds RARα or CAR heterodimerized with RXRα and appears to function synergistically with the IR-1 element to provide maximal induction of OSTβ in response to RA. These findings demonstrate a role for RARα and CAR in controlling OSTβ expression levels.

Background: Small ubiquitin-like modifiers (SUMO) are covalently conjugated to other proteins including nuclear receptors leading to modification of various cellular processes., Results: Ligand-dependent SUMOylation of farnesoid X receptor (FXR) negatively regulates the expression of its target genes., Conclusion: SUMO modification attenuates the capacity of FXR to function as a transcriptional activator., Significance: Defining post-translation modification of FXR bySUMOis important to understanding how this nuclear receptor functions in health and disease. The farnesoid X receptor (FXR) belongs to a family of ligand-activated transcription factors that regulate many aspects of metabolism including bile acid homeostasis. Here we show that FXR is covalently modified by the small ubiquitin-like modifier (Sumo1), an important regulator of cell signaling and transcription. Well conserved consensus sites at lysine 122 and 275 in the AF-1 and ligand binding domains, respectively, of FXR were subject to SUMOylation in vitro and in vivo. Chromatin immunoprecipitation (ChIP) analysis showed that Sumo1 was recruited to the bile salt export pump (BSEP), the small heterodimer partner (SHP), and the OSTα-OSTβ organic solute transporter loci in a ligand-dependent fashion. Sequential chromatin immunoprecipitation (ChIP-ReChIP) verified the concurrent binding of FXR and Sumo1 to the BSEP and SHP promoters. Overexpression of Sumo1 markedly decreased binding and/or recruitment of FXR to the BSEP and SHP promoters on ChIP-ReChIP. SUMOylation did not have an apparent effect on nuclear localization of FXR. Expression of Sumo1 markedly inhibited the ligand-dependent, transactivation of BSEP and SHP promoters by FXR/retinoid X receptor α (RXRα) in HepG2 cells. In contrast, mutations that abolished SUMOylation of FXR or siRNA knockdown of Sumo1 expression augmented the transactivation of BSEP and SHP promoters by FXR. Pathways for SUMOylation were significantly altered during obstructive cholestasis with differential Sumo1 recruitment to the promoters of FXR target genes. In conclusion, FXR is subject to SUMOylation that regulates its capacity to transactivate its target genes in normal liver and during obstructive cholestasis.

Base amino acid lysine residues play an important role in regulation of nuclear receptors [e.g., farnesyl X receptor (FXR)], leading to enhanced or suppressed biologic activity. To understand the molecular mechanisms and the subsequent effects in modulating FXR functions in diverse biologic processes, we individually replaced eight highly conserved lysine residues of human FXR (hFXR) with arginine. The effects of each mutated FXR on target gene activation, subcellular localization, protein-protein association, and protein-DNA interaction were investigated. Results demonstrated that K122R, K210R, K339R, and K460R mutants of hFXR significantly impaired target gene [organic solute transporter α/β and bile salt export pump (BSEP)] promoter reporter activity in a ligand-dependent fashion. None of the four mutants affected the nuclear localization of FXR. Protein interaction studies show that K210R slightly but significantly decreased FXR/retinoid X receptor (RXR) binding affinity but enhanced the interaction of FXR with lysine methyltransferase Set7/9 by ∼21%. K460R decreased the FXR interaction with Set7/9 by ∼45% but had no significant effects on interaction with RXR. Electrophoretic mobility shift assays demonstrated that hFXR-K210R and -K339R reduced the protein-DNA (IR1 element at hBSEP promoter) binding affinity by ∼80 and ∼90%, respectively. Computational-based protein modeling studies were consistent with these results and provided further insights into the potential underlying mechanisms responsible for these results. In conclusion, four highly conserved lysine residues of hFXR, K122, K210, K339, and K460, have been identified that play a critical role in FXR target gene regulation and molecular interaction (protein-protein and protein-DNA).

Background: Anterior capsular contraction syndrome is a potential complication of continuous curvilinear capsulorhexis (CCC). Three neodymium: yttrium-aluminum-garnet (Nd:YAG) laser relaxing incisions decrease anterior capsular contraction but the mechanism is unknown. The present study analyzed the biomechanical mechanism of three Nd:YAG laser relaxing incisions made to reduce anterior capsular contraction., Methods: A three-dimensional control model and a three-dimensional Nd:YAG model of the anterior capsule with an opening diameter of 6 mm were created. Three incisions of 1 mm in length were made centrifugally at intervals of 120° around the opening circle. The stress alterations of the anterior capsule after CCC with and without Nd:YAG relaxation were numerically simulated and compared., Results: In the control model, the stress was axially uniform in the inner area and relatively high near the inner rim of the opening. Meanwhile, in the Nd:YAG model, the stress level was very low in the inner opening areas, especially near the three incisions. The relaxing incisions in the Nd:YAG model significantly released the relatively high stress on the anterior capsule. Additionally, there was a high stress gradient near the relaxing incisions., Conclusion: Biomechanical effects of stress release may be the preventive mechanism of Nd:YAG incision against anterior capsular contraction syndrome.

The human organic solute transporter (hOST) is a heterodimer composed of alpha and beta subunits. Physical association of hOSTα and β subunits is essential for their polarized basolateral plasma membrane localization and function in the export of bile acids and steroids. To understand the role of carboxyl- and amino-tails of OSTβ and mechanisms underlying membrane localization of hOST, the effects of tail deletion of the hOSTβ subunit and biological reagents on membrane distribution and transport function of hOST were investigated in stably transfected MDCK cells. After deletion of 35 amino acids from the amino-tail of hOSTβ, the efflux transport activity and polarized membrane distribution of the truncated hOSTβ was abolished. A co-immunoprecipitation study verified that the amino-tail of hOSTβ is essential for the association with hOSTα subunit. Treatments with acytochalasin D (interrupting ctin-filaments), bafilomycin A1 (inhibiting vacuolar H(+)-ATPase), brefeldin A (disrupting the Golgi complex), and calphostin C (inhibiting protein kinase C), significantly disrupted the polarized membrane distribution of hOST and markedly reduced transport activity in stably transfected MDCK cells. In summary, the 35 amino acid amino-terminal fragment of hOSTβ contains critical information for interaction with the hOSTα subunit and subsequent trafficking to the plasma membrane. These studies suggest that the membrane sorting process of hOST is mediated by a bafilomycin A1-sensitive vesicular pathway that is associated with the actin-cytoskeleton network. The membrane localization of hOST is also partially mediated through a brefeldin A sensitive mechanism, which controls its transit from the ER to Golgi and is regulated by PKC.

Taking Chiping County in the Yellow River alluvial plain of Shandong Province as study area, a systematical survey was conducted on the 20 parameters of farmland soil physical and chemical properties under wheat/corn rotation, open air vegetable planting, sunlight greenhouse vegetable planting, and plastic shed vegetable planting, aimed to evaluate the effects of different planting patterns on the farmland soil quality in the plain. Significant differences (P < 0.05 or P < 0.01) were observed in the soil pH, soil moisture content, and the contents of soil organic matter, N, P, K, available S and Zn, and total salt under different planting patterns. The soil available P under all tested planting patterns and the soil available S under sunlight greenhouse vegetable planting presented a comparatively higher variability. Different planting patterns had significant effects on the soil quality, with the trend of protected vegetable planting > open air vegetable planting > wheat/ corn rotation. The effects were higher on soil chemical properties than on soil physical properties, and higher on soil organic matter and macronutrients than on soil secondary nutrients. Soil micronutrients were less affected. The main causes for these were the straw-returning of wheat and corn, the application of sulfur-containing and zinc fertilizers, and the long-term high rate fertilization of protected vegetable planting.

Unlabelled: Prior loss-of-function analyses revealed that ATPase class I type 8B member 1 [familial intrahepatic cholestasis 1 (FIC1)] posttranslationally activated the farnesoid X receptor (FXR). Mechanisms underlying this regulation were examined by gain-of-function studies in UPS cells, which lack endogenous FIC1 expression. FXR function was assayed in response to wild-type and mutated FIC1 expression constructs with a human bile salt export pump (BSEP) promoter and a variety of cellular localization techniques. FIC1 overexpression led to enhanced phosphorylation and nuclear localization of FXR that was associated with FXR-dependent activation of the BSEP promoter. The FIC1 effect was lost after mutation of the FXR response element in the BSEP promoter. Despite similar levels of FIC1 protein expression, Byler disease FIC1 mutants did not activate BSEP, whereas benign recurrent intrahepatic cholestasis mutants partially activated BSEP. The FIC1 effect was dependent on the presence of the FXR ligand, chenodeoxycholic acid. The effect of FIC1 on FXR phosphorylation and nuclear localization and its effects on BSEP promoter activity could be blocked with protein kinase C zeta (PKC zeta) inhibitors (pseudosubstrate or small interfering RNA silencing). Recombinant PKC zeta directly phosphorylated immunoprecipitated FXR. The mutation of threonine 442 of FXR to alanine yielded a dominant negative protein, whereas the phosphomimetic conversion to glutamate resulted in FXR with enhanced activity and nuclear localization. Inhibition of PKC zeta in Caco-2 cells resulted in activation of the human apical sodium-dependent bile acid transporter promoter., Conclusion: These results demonstrate that FIC1 signals to FXR via PKC zeta. FIC1-related liver disease is likely related to downstream effects of FXR on bile acid homeostasis. Benign recurrent intrahepatic cholestasis emanates from a partially functional FIC1 protein. Phosphorylation of FXR is an important mechanism for regulating its activity.

Introduction: The human organic anion transporting polypeptide C (OATPC) is one of the major transport proteins involved in the enterohepatic circulation of bile salts and plays an important role in vectorial transport of organic anions and drugs across hepatocytes., Materials and Methods: In this study, the effects of biological reagents on the membrane localization of OATPC were investigated by confocal microscopy and estrone-3-sulfate transport., Results: Our results demonstrated that the functional membrane expression of fluorescent chimera OATPC-GFP was achieved in non-polarized (COS7 and HEK293) and polarized (MDCK) cells. Both brefeldin A (a Golgi complex disruptor) and bafilomycin A1 (an inhibitor of vacuolar H+-ATPase) treatment significantly decreased the polarized membrane trafficking and markedly reduced the uptake of estrone-3-sulfate ( approximately 40-90%) in OATPC-GFP transfected cells, suggesting that membrane sorting of hOATPC-GFP was mediated by Golgi complex and vacuolar H+-ATPase-related vesicle transport pathways. Treatment with 8-Br-cAMP (a cAMP analog) stimulated OATPC-GFP membrane localization and enhanced estrone-3-sulfate uptake by approximately 20%. The protein kinase A (PKA) inhibitors (H89 and KT5720), but not a PKG inhibitor, blocked the polarized membrane expression of OATPC-GFP and reduced estrone-3-sulfate transport activity. The simultaneous treatment of cells with PKA activator/inhibitor and bafilomycin A1 demonstrated that bafilomycin A1 did not change the effects of 8-Br-cAMP and H89 on the membrane localization of OATPC-GFP compared with the use of 8-Br-cAMP and H89 alone., Discussion: These data suggest that a cAMP-PKA sensitive membrane sorting pathway for OATPC-GFP is independent of the vacuolar H+-ATPase associated (bafilomycin A1 sensitive) vesicle mediated membrane sorting pathway. In contrast, with combined treatment with brefeldin A, neither the PKA-activator (8-Br-cAMP) nor the inhibitor (H89) further altered the plasma membrane expression and transport activity of OATPC-GFP compared with brefeldin A treatment alone. These data suggest that the cAMP-PKA regulation of OATPC membrane expression involves the Golgi complex. When the Golgi apparatus was disrupted by brefeldin A treatment, the effects of cAMP-PKA on the Golgi-to-basolateral surface sorting process of OATPC was also diminished. In summary, the plasma membrane localization of human OATPC is mediated by Golgi complex and vacuolar H+-ATPase vesicle mediated membrane sorting pathways. cAMP-PKA regulates sorting process through the Golgi complex but not the vacuolar H+-ATPase associated vesicular pathway.

High-affinity, sodium-dependent dicarboxylate transporter (NaDC3) is responsible for transport of Krebs cycle intermediates and may involve in regulation of aging and life span. Hydropathy analysis predicts that NaDC3 contains 11 or 12 hydrophobic transmembrane (TM) domains. However, the actual membrane topological structure of NaDC3 remains unknown. In this study, confocal immunofluorescence microscopy and membrane biotinylation of epitope-tagged N and C termini of NaDC3 provide evidence of an extracellular C terminus and an intracellular N terminus, indicating an odd number of transmembrane regions. The position of hydrophilic loops within NaDC3 was identified with antibodies against the loops domains combined with cysteine accessibility methods. A confocal image of membrane localization and transport activity assay of the cysteine insertion mutants show behavior similar to that of wild-type NaDC3 in transfected HEK293 cells, suggesting that these mutants retain a native protein configuration. We find that NaDC3 contains 11 transmembrane helices. The loops 1, 3, 5, 7, and 9 face the extracellular side, and loops 2, 4, 6, and 10 face the cytoplasmic side. A re-entrant loop-like structure between TM8 and TM9 may protrude into the membrane. Our results support the topography of 11 transmembrane domains with an extracellular C terminus and an intracellular N terminus of NaDC3, and for the first time provide experimental evidence for a novel topological model for NaDC3.

Two proteins that mediate bile acid export from the ileal enterocyte, organic solute transporter (OST)-alpha and -beta, have recently been identified. It is unclear whether these two proteins associate directly and how they interact to mediate transport function and membrane localization. In this study, the protein-protein interactions, transport functions, and membrane localization of human (h)OST-alpha and -beta proteins were examined. The results demonstrated that coexpression of hOST-alpha and -beta in transfected cells resulted in a three- to fivefold increase of the initial rate of taurocholate influx or efflux compared with cells expressing each protein individually and nontransfected cells. Confocal microscopy demonstrated plasma membrane colocalization of hOST-alpha and -beta proteins in cells cotransfected with hOST-alpha and -beta cDNAs. Protein-protein interactions between hOST-alpha and -beta were demonstrated by mammalian two-hybrid and coimmunoprecipitation analyses. Truncation of the amino-terminal 50 amino acid extracellular residues of hOST-alpha abolished its interaction with hOST-beta and led to an intracellular accumulation of the two proteins and to only background levels of taurocholate transport. In contrast, carboxyl-terminal 28 amino acid truncated hOST-alpha still interacted with hOST-beta, and majority of this cytoplasmic tail-truncated protein was expressed on the basolateral membrane when it was stably cotransfected with hOST-beta protein in Madin-Darby canine kidney cells. In summary, hOST-alpha and -beta proteins are physically associated. The intracellular carboxyl-terminal domain of hOST-alpha is not essential for this interaction with hOST-beta. The extracellular amino-terminal fragment of hOST-alpha may contain important information for the assembly of the heterodimer and trafficking to the plasma membrane.

Human organic anion transporter hOAT1 plays critical roles in the body disposition of environmental toxins and clinically important drugs. In the present study, we examined the role of the C terminus of hOAT1 in its function. Combined approaches of cell surface biotinylation and transport analysis were employed for such purposes. It was found that deletion of the last 15 amino acids (residues 536-550) or the last 30 amino acids (residues 521-550) had no significant effect on transport activity. However, deletion of the entire C terminus (residues 506-550) completely abolished transport activity. Alanine scanning mutagenesis within the region of amino acids 506-520 led to the discovery of two critical amino acids: Glu-506 and Leu-512. Substitution of negatively charged Glu-506 with neutral amino acids alanine or glutamine resulted in complete loss of transport activity. However, such loss of transport activity could be rescued by substitution of Glu-506 with another negatively charged amino acid aspartic acid, suggesting the importance of negative charge at this position for maintaining the correct tertiary structure of the transporter, possibly by forming a salt bridge with a positively charged amino acid. Substitution of Leu-512 with amino acids carrying progressively smaller side chains including isoleucine, valine, and alanine resulted in mutants (L512I, L512V, and L512A) with increasingly impaired transport activity. However, the cell surface expression of these mutants was not affected. Kinetic analysis of mutant L512V revealed that the reduced transport activity of this mutant resulted mainly from a reduced maximum transport velocity Vmax without affecting the binding affinity (1/Km) of the transporter for its substrates, suggesting that the size of the side chain at position 512 critically affects transporter turnover number. Together, our results are the first to highlight the central role of the C terminus of hOAT1 in the function of this transporter.

The chloroplast HSP100/ClpB is a newly documented member of the ClpB family, but little was known about its role in imparting thermotolerance to cells. A cDNA coding for a HSP100/ClpB homolog has been cloned from Lycopersicon esculentum and termed as Lehsp100/ClpB (the cDNA sequence of Lehsp100/ClpB has been submitted to the GenBank database under accession number: AB219939). The protein encoded by the cDNA was most similar to the putative chloroplast HSP100/ClpBs in higher plants and the ClpB from Cyanobacterium Synechococcus sp. A 97 kDa protein, which matched the predicted size of mature LeHSP100/ClpB, was immunologically detected in chloroplast isolated from heat-treated tomato plants. In addition, the fusion protein, combining the transit sequence of LeHSP100/ClpB and GFP, was found to be located in chloroplast based on the observations of fluorescent microscope images. These results indicated the chloroplast-localization of LeHSP100/ClpB. Both the transcript and the protein of Lehsp100/ClpB were not detected under normal growth conditions, but they were induced by increasingly higher temperatures. An antisense Lehsp100/ClpB cDNA fragment was introduced into the tomato by Agrobacterium-mediated transformation. Antisense lines exhibited an extreme repression of heat-induced expression of Lehsp100/ClpB. The levels of chloroplast HSP60 and small HSP in antisense lines were identical to those of the control plants. After plants preconditioned at 38 degrees C for 2 h were exposed to a lethal heat shock at 46 degrees C for 2 h, the antisense lines were greatly impaired and withered in 21 days of the recovery phase, whereas the untransformed control plants and the vector-transformed plants survived. Furthermore, chlorophyll fluorescence measurements showed that PS II in antisense lines were more susceptible to the thermal irreversible inactivation than the untransformed and vector-transformed control plants. This work provides the first example that induction of chloroplast LeHSP100/ClpB contributes to the acquisition of thermotolerance in higher plants.

Small heat shock proteins (sHSPs) are the major family of HSP induced by heat stress in plants. In this report, an approximately 1.9kb of Lehsp23.8 5'-flanking sequence was isolated from tomato genome. By using the β-glucuronidase (GUS) reporter gene system, the developmental and tissue specific expression of the gus gene controlled by the Lehsp23.8 promoter was characterized in transgenic tomato plants. Strong GUS staining was detected in the roots, leaves, flowers, fruits and germinated seeds after heat shock. The heat-induced GUS activity was different in the floral tissues at various developmental stages. Fluorometric GUS assay showed that the heat-induced GUS activity was higher in the pericarp than in the placenta, and it was the lowest in the locular gel. The heat-shock induction of the Lehsp23.8 promoter depended on the different stages of fruit development. The optimal heat-shock temperatures leading to the maximal GUS activity in the pericarp of green, breaker, pink and red fruits were 42, 36, 39 and 39°C, respectively. The heat-induced GUS activity in tomato fruits increased gradually within 48h of treatment and weakened during tomato fruit ripening. Obvious GUS activities under cold, exogenous ABA and heavy metal (Cd(2+), Cu(2+), Pb(2+) or Zn(2+)) stress conditions were also detected. These results show that the Lehsp23.8 promoter is characterized as strongly heat-inducible and multiple-stress responsive., (Copyright © 2006 Elsevier Ireland Ltd. All rights reserved.)

The mechanisms underlying the transport of bile acids by apical sodium-dependent bile acid transporter (Asbt) are not well defined. To further identify the functionally relevant residues, thirteen conserved negatively (Asp and Glu) and positively (Lys and Arg) charged residues plus Cys-270 of rat Asbt were replaced with Ala or Gln by site-directed mutagenesis. Seven of the fourteen residues of rat Asbt were identified as functionally important by taurocholate transport studies, substrate inhibition assays, confocal microscopy, and electrophysiological methods. The results showed that Asp-122, Lys-191, Lys-225, Lys-256, Glu-261, and Lys-312,Lys-313 residues of rat Asbt are critical for transport function and may determine substrate specificity. Arg-64 may be located at a different binding site to assist in interaction with non-bile acid organic anions. For bile acid transport by Asbt, Na(+) ion movement is a voltage-dependent process that tightly companied with taurocholate movement. Asp-122 and Glu-261 play a critical role in the interaction of a Na(+) ion and ligand with Asbt. Cys-270 is not essential for the transport process. These studies provide new details about the amino acid residues of Asbt involved in binding and transport of bile acids and Na(+).

The full-length 2213-bp ftsH (filamentation temperature-sensitive H) cDNA was cloned from the cDNA library of heat-shocked tomato leaves. According to an open reading frame of 2019-bp, the deduced protein precursor was predicted to target chloroplast. The putative AAA (ATPases associated with diverse cellular activities) domain and the Zn(2+)-binding domain, characteristic of FtsH metalloproteases family, were found in the FtsH-like protein. Most similar to the FtsH6 of Arabidopsis thaliana, the tomato ftsH-like gene was named as Lycopersicon esculentum filamentation temperature-sensitive H6 (LeftsH6). Purified FtsH degraded casein but not BSA in vitro, whereas a FtsH mutant with the Glu(472) in the zinc-binding motif replaced by Gln had lost the protease activity. A single copy of LeftsH6 was detected in tomato genome by Southern blot analysis. Northern and Western blot analyses revealed consistently the heat-inducible character of the LeftsH6 gene. No LeftsH6 expression was detected after cold, salt, drought or light stress. The results provided the first experimental evidence of the existence of heat-inducible ftsH gene in higher plants.

The heat shock protein ClpB is a member of the Clp family and functions as molecular chaperones. ClpB is related to the acquired thermotolerance in organisms. A cDNA of 3144 bp was screened out of a tomato cDNA library. The polypeptide deduced from the longest ORF contains 980 amino acid residues, and was classified into HSP100/ClpB family based on the result of molecular phylogenesis analysis. Thus it was named as LeHSP110/ClpB according to its calculated molecular weight. LeHSP110/ClpB was characteristic of heat-inducibility but no constitutive expression, and was demonstrated to locate in chloroplastic stroma. An antisense cDNA fragment of LeHsp110/ClpB under the control of CaMV 35S promoter was introduced into tomato by Agrobacterium tumefactions-mediated method. At high temperature, the mRNA levels of LeHsp110/ClpB in antisense transgenic plants were lower than those in control plants. The PS II of transgenic plants is more sensitive to high temperature than that of control plants according to data of Fv/Fm. These results clearly showed that HSP110/ClpB plays an important role in thermotolerance of high plants.

The rat ileal apical sodium-dependent bile acid transporter (Asbt) transports conjugated bile acids in a Na+-dependent fashion and localizes specifically to the apical surface of ileal enterocytes. The mechanisms that target organic anion transporters to different domains of the ileal enterocyte plasma membrane have not been well defined. Previous studies (Sung, A.-Q., Arresa, M. A., Zeng, L., Swaby, I'K., Zhou, M. M., and Suchy, F. J. (2001) J. Biol. Chem. 276, 6825-6833) from our laboratory demonstrated that rat Asbt follows an apical sorting pathway that is brefeldin A-sensitive and insensitive to protein glycosylation, monensin treatment, and low temperature shift. Furthermore, a 14-mer signal sequence that adopts a beta-turn conformation is required for apical localization of rat Asbt. In this study, a vacuolar proton pump subunit (VPP-c, the 16-kDa subunit c of vacuolar H+-ATPase) has been identified as an interacting partner of Asbt by a bacterial two-hybrid screen. A direct protein-protein interaction between Asbt and VPP-c was confirmed in an in vitro pull-down assay and in an in vivo mammalian two-hybrid analysis. Indirect immunofluorescence confocal microscopy demonstrated that the Asbt and VPP-c colocalized in transfected COS-7 and MDCK cells. Moreover, bafilomycin A1 (a specific inhibitor of VPP) interrupted the colocalization of Asbt and VPP-c. A taurocholate influx assay and membrane biotinylation analysis showed that treatment with bafilomycin A1 resulted in a significant decrease in bile acid transport activity and the apical membrane localization of Asbt in transfected cells. Thus, these results suggest that the apical membrane localization of Asbt is mediated in part by the vacuolar proton pump associated apical sorting machinery.

The rat ileal sodium-dependent bile acid transporter (Asbt) is a polytopic membrane glycoprotein, which is specifically expressed on the apical domain of the ileal brush-border membrane. In the present study, an essential 14-amino acid (aa 335-348) sorting signal was defined on the cytoplasmic tail of Asbt with two potential phosphorylation sites motifs for casein kinase II ((335)SFQE) and protein kinase C (PKC) ((339)TNK). Two-dimension NMR spectra analysis demonstrated that a tetramer, (340)NKGF, which overlaps with the potential PKC site within the 14-mer signal sequence, adopts a type I beta-turn conformation. Replacement of the potential phosphorylation residue Ser(335) and Thr(339) with alanine or deletion of either the 4 ((335)SFQE) or 10 aa (338-348, containing (339)TNKGF) from the C terminus of Asbt resulted in a significantly decreased initial bile acid transport activity and increased the basolateral distribution of the mutants by 2-3-fold compared with that of wild type Asbt. Deletion of the entire last 14 amino acids (335-348) from the C terminus of Asbt abolished the apical expression of the truncated Asbt. Moreover, replacement of the cytoplasmic tail of the liver basolateral membrane protein, Na(+)/taurocholate cotransporting polypeptide, with the 14-mer peptide tail of Asbt redirected the chimera to the apical domain. In contrast, a chimera consisting of the 14-mer peptide of Asbt fused with green fluorescent protein was expressed in an intracellular transport vesicle-like distribution in transfected Madin-Darby canine kidney and COS 7 cells. This suggests that the apical localization of the 14-mer peptide requires a membrane anchor to support proper targeting. The results from biological reagent treatment and low temperature shift (20 degrees C) suggests that Asbt follows a transport vesicle-mediated apical sorting pathway that is brefeldin A-sensitive and insensitive to protein glycosylation, monensin treatment, and low temperature shift.

Background & Aims: Ileal malabsorption of bile salts is observed in Crohn's ileitis. We define the transcriptional mechanisms involved in cytokine-mediated repression of the rat apical sodium-dependent bile acid transporter (ASBT)., Methods: ASBT regulation was studied in IL-1beta-treated IEC-6 and Caco-2 cells and in indomethacin-treated rats., Results: Indomethacin-induced ileitis in Lewis rats leads to specific reductions in ileal ASBT messenger RNA and protein levels, whereas c-jun and c-fos are induced. The proinflammatory cytokines interleukin-1beta and tumor necrosis factor repress the activity of the ASBT promoter in Caco-2 and intestinal epithelial cell-6 cells. This effect is blocked by the proteasome inhibitor, MG-132, or by the phosphatidyl inositol 3-kinase inhibitor, wortmannin. Indomethacin (in vivo) or proinflammatory cytokine (in vitro) treatment leads to serine phosphorylation and nuclear translocation of c-fos. Mutation of a 5' activated protein (AP)-1 site inactivates the ASBT promoter, whereas mutation of the 3' site abrogates the proinflammatory cytokine-mediated repression. The 5' site binds a c-jun homodimer, whereas the 3' site binds a c-jun/c-fos heterodimer. c-Jun overexpression enhances ASBT promoter activity, whereas a dominant negative c-jun construct inactivates the promoter. c-Fos overexpression represses promoter activity. A 27 base pair cis-element from the 3' site in the ASBT promoter imparts cytokine-mediated down-regulation to a heterologous SV40 promoter construct., Conclusions: The ASBT promoter contains 2 distinct cis AP-1 elements; the 5' element binds homodimeric c-jun and mediates basal transcription. Inflammation is associated with up-regulation, phosphorylation, and nuclear translocation of c-fos, which then represses ASBT promoter activity via binding of the 3' AP-1 element by a c-fos/c-jun heterodimer.

Background & Aims: The mechanism by which cholelithiasis increases the risk of acute pancreatitis remains obscure. Because bile acids can enter the pancreas either by luminal diffusion or by interstitial leakage during gallstone impaction and pancreatitis is associated with impaired Ca(2+) signaling, we examined the effect of bile acids on pancreatic acinar cell signaling and the associated intracellular events., Methods: Rat pancreatic acinar cells were isolated by collagenase digestion and the effects of bile acids on [Ca(2+)](i) signaling, cell survival, inflammatory signals, and the molecular and functional expressions of bile uptake transporters were analyzed., Results: Bile acids specifically inhibited the sarco/endoplasmic reticulum Ca(2+) ATPase pump to chronically deplete part of the Ca(2+) stored in the endoplasmic reticulum. This in turn led to the activation of capacitative Ca(2+) entry and a chronic [Ca(2+)](i) load. The increase in [Ca(2+)](i) and Ca(2+) load activated the inflammation-associated signals of c-Jun amino-terminal kinases and NF-kappaB and led to cell death, which was inhibited by buffering [Ca(2+)](i) with 1,2-bis(2-aminophenoxy)ethane-N,N,N,N'-tetraacetic acid. A comprehensive molecular analysis of bile acid transporters revealed that pancreatic acinar cells express the bile uptake transporters Na(+)-taurocholate co-transporting polypeptide and organic anion transporting polypeptide in the luminal and basolateral membranes, respectively. Bile acid uptake into acinar cells was in part Na(+)-dependent and in part Na(+)-independent, suggesting that both transporters contribute to bile acid influx into acinar cells., Conclusions: These results suggest that bile acids can be transported into pancreatic acinar cells through specific membrane transporters and induce cell death by impairing cellular Ca(2+) signaling.

The human Na(+)-taurocholate cotransporting polypeptide (Ntcp) is located exclusively on the basolateral membrane of hepatocyte, but the mechanisms underlying its membrane sorting domain have not been fully elucidated. In the present study, a green fluorescent protein-fused human NTCP (NTCP-GFP) was constructed using the polymerase chain reaction and was stably transfected into Madin-Darby canine kidney (MDCK) and Caco-2 cells. Taurocholate uptake studies and confocal microscopy demonstrated that the polarity of basolateral surface expression of NTCP-GFP was maintained in MDCK cells but was lost in Caco-2 cells. Nocodazole (33 microM), an agent that causes microtubular depolymerization, partially disrupted the basolateral localization of NTCP-GFP by increasing apical surface expression to 33.5% compared with untreated cells (P < 0.05). Brefeldin A (BFA; 1-2 microM) disrupted the polarized basolateral localization of NTCP, but monensin (1.4 microM) had no affect on NTCP-GFP localization. In addition, low-temperature shift (20 degrees C) did not affect the polarized basolateral surface sorting of NTCP-GFP and repolarization of this protein after BFA interruption. In summary, these data suggest that the polarized basolateral localization of human NTCP is cell specific and is mediated by a novel sorting pathway that is BFA sensitive and monensin and low-temperature shift insensitive. The process may also involve microtubule motors.

To understand the potential functions of the cytoplasmic tail of Na(+)/taurocholate cotransporter (Ntcp) and to determine the basolateral sorting mechanisms for this transporter, green fluorescent protein-fused wild type and mutant rat Ntcps were constructed and the transport properties and cellular localization were assessed in transfected COS 7 and Madin-Darby canine kidney (MDCK) cells. Truncation of the 56-amino acid cytoplasmic tail demonstrates that the cytoplasmic tail of rat Ntcp is involved membrane delivery of this protein in nonpolarized and polarized cells and removal of the tail does not affect the bile acid transport function of Ntcp. Using site-directed mutagenesis, two tyrosine residues, Tyr-321 and Tyr-307, in the cytoplasmic tail of Ntcp have been identified as important for the basolateral sorting of rat Ntcp in transfected MDCK cells. Tyr-321 appears to be the major basolateral-sorting determinant, and Tyr-307 acts as a supporting determinant to ensure delivery of the transporter to the basolateral surface, especially at high levels of protein expression. When the two Tyr-based basolateral sorting motifs have been removed, the N-linked carbohydrate groups direct the tyrosine to alanine mutants to the apical surface of transfected MDCK cells. The major basolateral sorting determinant Tyr-321 is within a novel beta-turn unfavorable tetrapeptide Y(321)KAA, which has not been found in any naturally occurring basolateral sorting motifs. Two-dimensional NMR spectroscopy of a 24-mer peptide corresponding to the sequence from Tyr-307 to Thr-330 on the cytoplasmic tail of Ntcp confirms that both the Tyr-321 and Tyr-307 regions do not adopt any turn structure. Since the major motif YKAA contains a beta-turn unfavorable structure, the Ntcp basolateral sorting may not be related to the clathrin-adaptor complex pathway, as is the case for many basolateral proteins.

The rat ileal apical Na+-dependent bile acid transporter (ASBT) and the liver Na+-taurocholate cotransporting polypeptide (Ntcp) are members of a new family of anion transporters. These transport proteins share limited sequence homology and almost identical predicted secondary structures but are localized to the apical surface of ileal enterocytes and the sinusoidal surface of hepatocytes, respectively. Stably transfected Madin-Darby canine kidney (MDCK) cells appropriately localized wild-type ASBT and Ntcp apically and basolaterally as assessed by functional activity and immunocytochemical localization studies. Truncated and chimeric transporters were used to determine the functional importance of the cytoplasmic tail in bile acid transport activity and membrane localization. Two cDNAs were created encoding a truncated transporter in which the 56-amino-acid COOH-terminal tail of Ntcp was removed or substituted with an eight-amino-acid epitope FLAG. For both mutants there was some loss of fidelity in basolateral sorting in that approximately 75% of each protein was delivered to the basolateral surface compared with approximately 90% of the wild-type Ntcp protein. In contrast, deletion of the cytoplasmic tail of ASBT led to complete loss of transport activity and sorting to the apical membrane. An Ntcp chimera in which the 56-amino-acid COOH-terminal tail of Ntcp was replaced with the 40-amino-acid cytoplasmic tail of ASBT was largely redirected (82.4 +/- 3.9%) to the apical domain of stably transfected MDCK cells, based on polarity of bile acid transport activity and localization by confocal immunofluorescence microscopy. These results indicate that a predominant signal for sorting of the Ntcp protein to the basolateral domain is located in a region outside of the cytoplasmic tail. These studies have further shown that a novel apical sorting signal is localized to the cytoplasmic tail of ASBT and that it is transferable and capable of redirecting a protein normally sorted to the basolateral surface to the apical domain of MDCK cells.