Ectopic expression of UGT84A2 delayed flowering by indole-3-butyric acid-mediated transcriptional repression of ARF6 and ARF8 genes in Arabidopsis.

KEY MESSAGE: Ectopic expression of auxin glycosyltransferase UGT84A2 in Arabidopsis can delay flowering through increased indole-3-butyric acid and suppressed transcription of ARF6, ARF8 and flowering-related genes FT, SOC1, AP1 and LFY. Auxins are critical regulators for plant growth and developmental processes. Auxin homeostasis is thus an important issue for plant biology. Here, we identified an indole-3-butyric acid (IBA)-specific glycosyltransferase, UGT84A2, and characterized its role in Arabidopsis flowering development. UGT84A2 could catalyze the glycosylation of IBA, but not indole-3-acetic acid (IAA). UGT84A2 transcription expression was clearly induced by IBA. When ectopically expressing in Arabidopsis, UGT84A2 caused obvious delay in flowering. Correspondingly, the increase of IBA level, the down-regulation of AUXIN RESPONSE FACTOR 6 (ARF6) and ARF8, and the down-regulation of flowering-related genes such as FLOWERING LOCUS T (FT), SUPPRESSOR OF OVEREXPRESSION OF CO1(SOC1), APETALA1 (AP1), and LEAFY(LFY) were observed in transgenic plants. When exogenously applying IBA to wild-type plants, the late flowering phenotype, the down-regulation of ARF6, ARF8 and flowering-related genes recurred. We examined the arf6arf8 double mutants and found that the expression of flowering-related genes was also substantially decreased in these mutants. Together, our results suggest that glycosyltransferase UGT84A2 may be involved in flowering regulation through indole-3-butyric acid-mediated transcriptional repression of ARF6, ARF8 and downstream flowering pathway genes.

The Arabidopsis UDP-glycosyltransferases UGT79B2 and UGT79B3, contribute to cold, salt and drought stress tolerance via modulating anthocyanin accumulation.

The plant family 1 UDP-glycosyltransferases (UGTs) are the biggest GT family in plants, which are responsible for transferring sugar moieties onto a variety of small molecules, and control many metabolic processes; however, their physiological significance in planta is largely unknown. Here, we revealed that two Arabidopsis glycosyltransferase genes, UGT79B2 and UGT79B3, could be strongly induced by various abiotic stresses, including cold, salt and drought stresses. Overexpression of UGT79B2/B3 significantly enhanced plant tolerance to low temperatures as well as drought and salt stresses, whereas the ugt79b2/b3 double mutants generated by RNAi (RNA interference) and CRISPR-Cas9 strategies were more susceptible to adverse conditions. Interestingly, the expression of UGT79B2 and UGT79B3 is directly controlled by CBF1 (CRT/DRE-binding factor 1, also named DREB1B) in response to low temperatures. Furthermore, we identified the enzyme activities of UGT79B2/B3 in adding UDP-rhamnose to cyanidin and cyanidin 3-O-glucoside. Ectopic expression of UGT79B2/B3 significantly increased the anthocyanin accumulation, and enhanced the antioxidant activity in coping with abiotic stresses, whereas the ugt79b2/b3 double mutants showed reduced anthocyanin levels. When overexpressing UGT79B2/B3 in tt18 (transparent testa 18), a mutant that cannot synthesize anthocyanins, both genes fail to improve plant adaptation to stress. Taken together, we demonstrate that UGT79B2 and UGT79B3, identified as anthocyanin rhamnosyltransferases, are regulated by CBF1 and confer abiotic stress tolerance via modulating anthocyanin accumulation.

Ectopic expression of UGT75D1, a glycosyltransferase preferring indole-3-butyric acid, modulates cotyledon development and stress tolerance in seed germination of Arabidopsis thaliana.

The formation of auxin glucose conjugate is proposed to be one of the molecular modifications controlling auxin homeostasis. However, the involved mechanisms and relevant physiological significances are largely unknown or poorly understood. In this study, Arabidopsis UGT75D1 was at the first time identified to be an indole-3-butyric acid (IBA) preferring glycosyltransferase. Assessment of enzyme activity and IBA conjugates in transgenic plants ectopically expressing UGT75D1 indicated that the UGT75D1 catalytic specificity was maintained in planta. It was found that the expression pattern of UGT75D1 was specific in germinating seeds. Consistently, we found that transgenic seedlings with over-produced UGT75D1 exhibited smaller cotyledons and cotyledon epidermal cells than the wild type. In addition, UGT75D1 was found to be up-regulated under mannitol, salt and ABA treatments and the over-expression lines were tolerant to osmotic and salt stresses during germination, resulting in an increased germination rate. Quantitative RT-PCR analysis revealed that the mRNA levels of ABA INSENSITIVE 3 (ABI3) and ABI5 gene in ABA signaling were substantially down-regulated in the transgenic lines under stress treatments. Interestingly, AUXIN RESPONSE FACTOR 16 (ARF16) gene of transgenic lines was also dramatically down-regulated under the same stress conditions. Since ARF16 functions as an activator of ABI3 transcription, we supposed that UGT75D1 might play a role in stress tolerance during germination through modulating ARF16-ABI3 signaling. Taken together, our work indicated that, serving as the IBA preferring glycosyltransferase but distinct from other auxin glycosyltransferases identified so far, UGT75D1 might be a very important player mediating a crosstalk between cotyledon development and stress tolerance of germination at the early stage of plant growth.

Systematic analyses reveal uniqueness and origin of the CFEM domain in fungi.

CFEM domain commonly occurs in fungal extracellular membrane proteins. To provide insights for understanding putative functions of CFEM, we investigate the evolutionary dynamics of CFEM domains by systematic comparative genomic analyses among diverse animals, plants, and more than 100 fungal species, which are representative across the entire group of fungi. We here show that CFEM domain is unique to fungi. Experiments using tissue culture demonstrate that the CFEM-containing ESTs in some plants originate from endophytic fungi. We also find that CFEM domain does not occur in all fungi. Its single origin dates to the most recent common ancestors of Ascomycota and Basidiomycota, instead of multiple origins. Although the length and architecture of CFEM domains are relatively conserved, the domain-number varies significantly among different fungal species. In general, pathogenic fungi have a larger number of domains compared to other species. Domain-expansion across fungal genomes appears to be driven by domain duplication and gene duplication via recombination. These findings generate a clear evolutionary trajectory of CFEM domains and provide novel insights into the functional exchange of CFEM-containing proteins from cell-surface components to mediators in host-pathogen interactions.

Correlation of lower concentrations of hydrogen sulfide with atherosclerosis in chronic hemodialysis patients with diabetic nephropathy.

BACKGROUND/AIMS: To explore the relationship between hydrogen sulfide (H2S) and uremic accelerated atherosclerosis (UAAS) in chronic hemodialysis patients with diabetic nephropathy (CHD/DN). METHODS: A total of 36 CHD/DN and 32 chronic hemodialyzed non-diabetic patients with chronic glomerulonephritis (CHD/non-DN) were studied. Plasma H2S was measured with a sulfide sensitive electrode. RESULTS: Plasma H2S in CHD/DN was significantly lower than that in CHD/non-DN patients. Plasma H2S was positively correlated with plasma TGF-ß1, and negatively correlated with MMP-12 in CHD/DN patients. CHD/DN patients exhibited higher CCA-IMT, hsCRP, and lower H2S levels than in CHD/non-DN patients. Moreover, in CHD/DN patients, CCA-IMT was negatively correlated with plasma H2S, and positively correlated with hsCRP and LDL. On multiple regression analysis, H2S levels exhibited independent association with IMT in CHD/DN patients. CONCLUSIONS: These findings suggest possible linkage between H2S metabolism and TGF-ß/Smad signaling pathway modulation abnormalities that may contribute to the development of UAAS in CHD/DN patients.

UGT74D1 is a novel auxin glycosyltransferase from Arabidopsis thaliana.

Auxin is one type of phytohormones that plays important roles in nearly all aspects of plant growth and developmental processes. The glycosylation of auxins is considered to be an essential mechanism to control the level of active auxins. Thus, the identification of auxin glycosyltransferases is of great significance for further understanding the auxin regulation. In this study, we biochemically screened the group L of Arabidopsis thaliana glycosyltransferase superfamily for enzymatic activity toward auxins. UGT74D1 was identified to be a novel auxin glycosyltransferase. Through HPLC and LC-MS analysis of reaction products in vitro by testing eight substrates including auxins and other compounds, we found that UGT74D1 had a strong glucosylating activity toward indole-3-butyric acid [IBA], indole-3-propionic acid [IPA], indole-3-acetic acid [IAA] and naphthaleneacetic acid [NAA], catalyzing them to form corresponding glucose esters. Biochemical characterization showed that this enzyme had a maximum activity in HEPES buffer at pH 6.0 and 37°C. In addition, the enzymatic activity analysis of crude protein and the IBA metabolite analysis from transgenic Arabidopsis plants overexpressing UGT74D1 gene were also carried out. Experimental results indicated that over-production of the UGT74D1 in plants indeed led to increased level of the glucose conjugate of IBA. Moreover, UGT74D1 overexpression lines displayed curling leaf phenotype, suggesting a physiological role of UGT74D1 in affecting the activity of auxins. Our current data provide a new target gene for further genetic studies to understand the auxin regulation by glycosylation in plants.

[Association between the synovial expression of cyclic citrullinated peptide and susceptibility variants of HLA-DRB1 shared epitope alleles and PADI 4 gene single nucleotide polymorphisms in patients with rheumatoid arthritis].

OBJECTIVE: To analyze the association between the synovial expression of cyclic citrullinated peptide (CCP) and susceptibility variants of HLA-DRB1 shared epitope (SE) alleles and/or peptidylarginine deiminase 4 (PADI4) gene single nucleotide polymorphisms (SNP) in patients with rheumatoid arthritis (RA). METHODS: From October 2008 to December 2011, 53 RA patients and 42 controls were enrolled. The expression of CCP in RA synovial tissues was detected by immunohistochemical assay with 6×His tagged anti-CCP single chain fragment V (ScFv) antibodies generated by pHEN2 phagemid recombinant antibodies display system. PADI4 SNP was genotyped by reverse transcription cDNA sequencing and heterozygote was DNA haplotype was mapped by TA clone sequencing. HLA-DRB1 SE alleles were analyzed by sequence specific primer-polymerase chain reaction (SSP-PCR). RESULTS: The prevalence of synovial CCP expression was significantly different between RA group and the control (76.9% and 11.4% respectively, P < 0.01). The frequencies of 2 SNPs (PADI4_89 G+ and PADI4_104 T+) varied significantly between the groups(P < 0.05). Compared with the major haplotypes, only these two minor alleles were associated with the increased RA susceptibility (OR = 3.67 and 2.53, P < 0.05). SE+ alleles was strongly associated with RA susceptibility (OR = 5.57, P < 0.01). The synovial expression of CCP in RA was strongly associated with SE+ alleles, only 2 minor SNPs (PADI4_89 G+ and PADI4_104T+) and the combination. Serum anti-CCP titers were significantly associated with SE+ alleles, PADI4_104T+, SE+/PADI4_89 G+ and SE+/PADI4_104T+ haplotype. CONCLUSION: The synovial expression of CCP and the generation of anti-CCP antibodies are strongly associated with SE alleles and/or certain PADI4 gene SNP in RA.

Enrichment of a common wheat genetic map and QTL mapping for fatty acid content in grain.

DArT and SSR markers were used to saturate and improve a previous genetic map of RILs derived from the cross Chuan35050 × Shannong483. The new map comprised 719 loci, 561 of which were located on specific chromosomes, giving a total map length of 4008.4 cM; the rest 158 loci were mapped to the most likely intervals. The average chromosome length was 190.9 cM and the marker density was 7.15 cM per marker interval. Among the 719 loci, the majority of marker loci were DArTs (361); the rest included 170 SSRs, 100 EST-SSRs, and 88 other molecular and biochemical loci. QTL mapping for fatty acid content in wheat grain was conducted in this study. Forty QTLs were detected in different environments, with single QTL explaining 3.6-58.1% of the phenotypic variations. These QTLs were distributed on 16 chromosomes. Twenty-two QTLs showed positive additive effects, with Chuan35050 increasing the QTL effects, whereas 18 QTLs were negative with increasing effects from Shannong483. Six sets of co-located QTLs for different traits occurred on chromosomes 1B, 1D, 2D, 5D, and 6B.

Hypocholesterolemic effects of Auricularia auricula ethanol extract in ICR mice fed a cholesterol-enriched diet.

The cholesterol-lowering properties of Auricularia auricula are commonly attributed to the presence of polysaccharides based on previous research. The present study was designed to investigate the effects of ethanol extract of A. auricula (AAE) on hypercholesterolemia in ICR mice. AAE contained more than 16% (g/g) polyphenolic compounds, excluding other interfering factors such as polysaccharides, water-soluble fibre and protein. Thirty-six mice were randomly assigned to three groups (n = 12). The experimental group was fed cholesterol-enriched diet (CED) with oral administration of AAE (150 mg/kg/d b.w.) for 8-week, normal control group and CED control group received either a regular diet (RD) or CED along with oral administration of equal volume distilled water. Serum lipid profiles and antioxidant status were measured in addition to fecal neutral cholesterol and bile acids. AAE showed a remarkable hypocholesterolemic effect, improving antioxidant status, decreasing the level of total cholesterol and atherosclerosis index, increasing the level of high-density lipoprotein cholesterol and fecal excretion of bile acids. No apparent effects on serum triglycerides, low-density lipoprotein cholesterol, fecal excretion of neutral cholesterol and feeding efficiency were observed among all groups. These results indicated that A. auricula functional components, which prevented hypercholesterolemia contained polyphenolic compounds, in addition to polysaccharides.

A comparative study of influential factors correlating with early and late hypothyroidism after (131)I therapy for Graves' disease.

BACKGROUND: (131)I therapy is recognized as the simplest, safest, least expensive, and most effective treatment, and accepted by more and more patients. However its curative effect is influenced by many factors, therefore there are some difficulties for doctors to establish individual treatment strategy. The aims of this study were to determine the incidence of early and late hypothyroidism after (131)I treatment for Graves' disease (GD) and to compare their correlation, to observe and analyze the influential factors and to understand the predictabilities of them. METHODS: Five hundred GD patients (144 males, 356 females; age (41.2 +/- 12.3) years) received (131)I treatment for the first time. The therapeutic procedure was carried out as the following: undergoing (131)I uptake test to obtain maximum of thyroid uptake value and effective half-life (EHL) time; estimating the thyroid's weight by ultrasonography; determination of thyroid hormones and correlative antibodies; pre-therapy physical examination; thyroid imaging; calculating (131)I therapeutic dosage; per os uptake of the determined (131)I dosage; follow-up appraisal of curative effect. The observing parameters included age, gender, thyroid weight, GD duration, condition of onset, state of disease, course of treatment, EHL time, maximum of thyroid uptake value, (131)I dosage and titer of correlative antibodies. We sorted out the data and used both univariate and multivariate analysis to evaluate them statistically. RESULTS: The incidence rates of early and late hypothyroidism were 33.2% and 6.6% respectively after (131)I treatment and approximately 22.2% cases of late hypothyroidism developed from early hypothyroidism. The influential factors of early hypothyroidism included course of GD, the highest thyroid uptake ratio of (131)I, EHL time and thyroid microsome antibody (TMAb), etc. A multivariate analysis on late hypothyroidism showed that female patients, with recurrence after anti-thyroid drug treatment and higher thyroid weight, had lower possibility of late hypothyroidism after (131)I therapy. CONCLUSIONS: The incidence of early hypothyroidism is higher than that of late hypothyroidism. The highest thyroid uptake ratio of (131)I, EHL and TMAb will increase the possibility of early hypothyroidism, while GD course is the protective factor. Higher (131)I dosage, longer EHL and higher TMAb titer will also increase the possibility of late hypothyroidism. The multi-perspective and multi-factor analysis has the benefit to establish individualized treatment strategy.

Thrombosis of the superior vena cava and auxiliary branches in patients with indwelling catheterization of the internal jugular vein.

BACKGROUND: Central venous thrombosis is a serious and life-threatening complication in hemodialysis (HD) patients with an indwelling catheter. The present study aimed to investigate the prevalence and characteristics of thrombosis of the superior vena cava and auxiliary branches in Chinese HD patients with an indwelling internal jugular venous catheter and to explore its risk factors. METHODS: Fifty-four patients on maintenance hemodialysis (MHD) with an indwelling catheter were enrolled in this cross-sectional study. The thrombosis of the internal jugular vein, subclavical vein, brachiocephalic vein and superior vena cava was assessed by vascular ultrasound. Collected were data on age, gender, ultrafiltration volume, Kt/V, blood pressure, levels of hemoglobin, serum albumin, lipid, calcium, and phosphorus, and parathyroid hormone. RESULTS: The patients were given short- or long-term double lumen central venous catheters. Among them, 42 patients had the catheter placed into the right internal jugular vein, and 12 patients into the left internal jugular vein. Different degrees of central venous thrombosis were found in 33 patients (61.1%). The prevalence of thrombosis in the jugular vein, brachiocephalic vein, subclavical vein and superior vena cava was 61.1% (33/54), 44.4% (24/54), 16.7% (9/54) and 5.6% (3/54), respectively. Among the 33 HD patients with central venous thrombosis, the percentages for one, two, three and four affected veins were 27.3% (9/33), 45.4% (15/33), 18.2% (6/33) and 9.1% (3/33), respectively. Twelve (12/33, 36.4%) of the 33 HD patients with central venous thrombosis had clinical symptoms. Nine patients (27.3%) had edema of the upper extremity and 3 (9.1%) had new-onset symptoms of pulmonary embolism such as cough, chest distress and short breath. The incidences of diabetes mellitus and malignant tumor and levels of lipoprotein a and homocysteic acid were significantly higher in the HD patients with central venous thrombosis than in those without central venous thrombosis. Logistic regressive analysis revealed that high level of homocysteic acid was the important risk factor for central venous thrombosis in HD patients with indwelling catheterization of the internal jugular vein. CONCLUSIONS: The prevalence of central venous thrombosis in Chinese HD patients with indwelling catheterization of the internal jugular vein is quite high, especially in those patients with diabetes mellitus, malignant tumor, high levels of serum lipoprotein and homocysteic acid. Its clinical symptoms are insidious but dangerous. High level of homocysteic acid may be the important risk factor for central venous thrombosis in Chinese HD patients with indwelling catheterization of the internal jugular vein.

[Effect of co-inhibition of MCT1 gene and NHE1 gene on proliferation and growth of human lung adenocarcinoma cells].

BACKGROUND & OBJECTIVE: The authors previously discovered that the intracellular pH values (pHi) of tumor cells could be decreased, which led tumor cells to acidify and die, when the expressions of the first subtype of monocarboxylate transporter (MCT1) gene and the first subtype of Na+/H+ exchanger (NHE1) gene in tumor cell membranes had been inhibited by each corresponding antisense gene respectively. However it was not clear whether there were co-effects on the pHi, proliferation, and growth of tumor cells, after two genes were inhibited at the same time. METHODS: pLXSN-MCT1 and pLXSN-NHE1, the corresponding antisense gene expression vectors of MCT1 and NHE1 genes, were introduced into human lung adenocarcinoma A549 cells at the same time with electroporation. The positive colonies were selected by G418. Using PCR and RT-PCR technique, it was confirmed that antisense genes of MCT1 and NHE1 genes integrated with A549 genomic DNA. The pHi and lactate content were detected with spectrophotometry. Cell growth cycle was measured with flow cytometer. The cells co-transfected antisense genes, cells transfected MCT1 antisense gene and A549 cells were respectively inoculated in nude mouse subcutaneous to observe the growth of transplantation tumors. RESULTS: Compared with A549 cells, the pHi of cells co-transfected antisense genes was remarkably decreased (P < 0.05), while lactate content was remarkably increased (P < 0.001). The cell cycle was blocked at G0-G1 period. And the transplantation tumors of nude mice inoculated co-transfected antisense gene-cells and inoculated MCT1-antisense-gene-transfected cells were significantly lighter and smaller than ones inoculated A549 cells (P < 0.001). CONCLUSION: MCT1 and NHE1 genes play important regulation roles in proliferation and growth of tumor cells, probably by affecting pHi.