Signaling related with biphasic effects of bisphenol A (BPA) on Sertoli cell proliferation: A comparative proteomic analysis

Background

Biphasic effects on cell proliferation of bisphenol A (BPA) can occur at lesser or greater exposures. Sertoli cells play a pivotal role in supporting proliferation and differentiation of germ cells. The mechanisms responsible for inverse effects of great and low concentrations of BPA on Sertoli cell proliferation need further study.

Methods

We utilized proteomic study to indentify the protein expression changes of Sertoli TM4 cells treated with 10^− 8 M and 10^− 5 M BPA. The further mechanisms related to mitochondria, energy metabolism and oxidative stress were investigated by qRT-PCR and Western-blotting analysis.

Results

Proteomic studies identified 36 proteins and two major clusters of proteins including energy metabolism and oxidative stress expressed with opposite changes in Sertoli cells treated with 10^− 8 M and 10^− 5 M BPA, respectively, for 24 h. Exposure to 10^− 5 M BPA resulted in greater oxidative stress and then inhibited cell proliferation, while ROS scavenger NAC effectively blocked these effects. Exposure to 10^− 8 M BPA caused higher intercellular ATP, greater activities of mitochondria, and resulted in significant proliferation of TM4 cells, while oligomycin A, an inhibitor of ATP synthase, abolished these growth advantages.

Conclusions

Our study demonstrated that micromolar BPA inhibits proliferation of Sertoli cells by elevating oxidative stress while nanomolar BPA stimulates proliferation by promoting energy metabolism.

General significance

Micromolar BPA inhibits cell proliferation by elevating oxidative stress while nanomolar BPA stimulates cell proliferation by promoting energy metabolism.     

Dual effects of estrogen on vascular smooth muscle cells: receptor-mediated proliferative vs. metabolite-induced pro-senescent actions

Objective

To investigate the mechanism for the dual effects of estrogen on vascular smooth muscle cells (VSMCs).

Methods

Cultured rat VSMCs were exposed to gradient concentrations (10⁻⁹-10⁻⁵ M) of 17β-estradiol (E₂) with or without pre-administration of a broad-spectrum CYP450 inhibitor 1-aminobenzotriazole (ABT) (10 × 10⁻⁶ M) and an estrogen receptor (ER) antagonist ICI 182,780 (10⁻⁶ M), respectively. The growth, cell cycle progression, premature senescence, estrogen metabolites, reactive oxygen species (ROS) and DNA damage of the cells were analyzed with cell counting assay, flow cytometry, Western blot, liquid chromatography-mass spectrometry and comet assay, respectively.

Results

E₂ in its physiological levels from 10⁻⁹ M to 10⁻⁸ M had a concentration-dependent promoting effect on growth of VSMCs. However, when the concentration increased over 10⁻⁸ M, the growth-promoting effect gradually reversed to a growth-inhibiting action. When the activity of CYP450s was blocked by ABT, the growth-promoting effect of E₂ increased and did not reverse at high concentrations. Whereas when the ERs were blocked by ICI 182,780, E₂ showed a pure growth-inhibiting effect. The E₂ metabolites 2- and 4-hydroxyestradiols accumulated with the increase of E₂ over 10⁻⁸ M, which accompanied by increased ROS, DNA damage and cellular senescence. All of these changes were eliminated by block of CYP450s, indicating that the VSMC growth inhibition by E₂ is due to an increased production of ROS from accumulated E₂ metabolites which induces DNA damage, leading to VSMC premature senescence.

Conclusion

The complex effect of E₂ is due to two opposite actions: one ER-mediated and proliferative, and the other estrogen metabolite-induced and pro-senescent.     

Phagocytic activity,respiratory burst,cytoplasmic free-Ca concentration and apoptotic cell ratio of haemocytes from the black tiger shrimp, Penaeus monodon under acute copper stress

The aim of this study was to investigate the cellular toxicity of copper-induced injury to the black tiger shrimp Penaeus monodon. The 24 h, 48 h, 72 h and 96 h LC₅₀ (median lethal concentration) of Cu²⁺ on P. monodon (11.63 ± 1.14 g) were found to be 3.49, 1.54, 0.73 and 0.40 mg L^− 1, respectively. Total haemocyte count (THC), phagocytic activity, respiratory burst (RB), cytoplasmic free-Ca²⁺ (cf-Ca²⁺) concentration and apoptotic cell ratio of shrimp were determined after exposure to different concentrations of Cu²⁺ (0, 0.05, 0.5, 1.5 and 3.5 mg L^− 1) for 0, 6, 12, 24 and 48 h. There was no significant effect on the analytic indicator of shrimp exposed to 0.05 mg L^− 1 Cu²⁺. THC decreased after Cu-exposure to 0.5 mg L^− 1 for 48 h, 1.5 mg L^− 1 for 24 h and 3.5 mg L^− 1 for 12 h. Phagocytic activity decreased in P. monodon following 48 h exposure to 3.5 mg L^− 1 Cu²⁺. RB was induced after 6 h exposure to 0.5, 1.5 and 3.5 mg L^− 1 Cu²⁺. cf-Ca²⁺ concentration increased after 48 h exposure to 0.5 mg L^− 1 Cu²⁺, and 12 h exposure to 1.5 and 3.5 mg L^− 1 Cu²⁺. The percentage of apoptotic cells increased to 9.5%, 16.3% and 18.6% respectively following 48 h exposure to 0.5, 1.5 and 3.5 mg L^− 1 Cu²⁺. These results indicate that Cu can induce oxidative stress, elevation of cf-Ca²⁺ and cell apoptosis, and inhibit phagocytic activity in the shrimp P. monodon, and the lethal injury of Cu²⁺ to P. monodon may be mainly due to the sharp reduction of THC caused by ROS-induced apoptosis.     

Evaluation of selenite bioremoval from liquid culture by Enterococcus species

The genus Enterococcus belong to the genera of bacteria that produce lactic acid and can confer health benefits to living organisms. Selenium (Se) is an essential micronutrient for humans and animals. Thirty-six Enterococcus species isolated from dairy products were screened for Se(IV) sorption capacity for use as a probiotics in animal nutrition. Several isolates grew luxuriantly and significantly removed Se(IV) from Se(IV) amended medium. Two isolates, LAB 14 and LAB 18, identified by 16S rRNA gene sequence analysis as Enterococcus faecalis (98% nucleotide sequence similarity) and Enterococcus faecium (97% nucleotide sequence similarity), respectively, were selected for further studies. The two isolates grew optimally and removed selenium at initial pH 7.0. Optimum removal of Se(IV) from the medium was recorded at 25 °C. Time course studies showed that after 8 h of incubation LAB 14 and LAB 18 cultures displayed the highest biomass production and Se(IV) bioremoval and most selenite in culture depleted in 24 h. At initial concentrations of 10 mg L⁻¹ and 60 mg L⁻¹, E. faecium (LAB 18) removed 9.91 mg L⁻¹ and 59.70 mg L⁻¹, respectively after 24 h. Similar Se(IV) bioremoval capacity was recorded with E. faecalis (LAB 14). Substantial amount of Se was detected in biomass of E. faecium (0.4599 mg g⁻¹ of dry weight) and E. faecalis (0.4759 mg g⁻¹ of dry weight). The significant uptake and transformation of Se(IV) by the Enterococcus species observed in this study suggest that they can be used to deliver dietary Se through feed augmentation with Se(IV)-enriched Enterococcus biomass.     

Fungal solid state fermentation on agro-industrial wastes for acid wastewater decolorization in a continuous flow packed-bed bioreactor

This study was aimed at developing a process of solid state fermentation (SSF) with the fungi Pleurotus ostreatus and Trametes versicolor on apple processing residues for wastewater decolorization. Both fungi were able to colonize apple residues without any addition of nutrients, material support or water. P. ostreatus produced the highest levels of laccases (up to 9 U g⁻¹ of dry matter) and xylanases (up to 80 U g⁻¹ of dry matter). A repeated batch decolorization experiment was set up with apple residues colonized by P. ostreatus, achieving 50% decolorization and 100% detoxification after 24 h, and, adding fresh wastewater every 24 h, a constant decolorization of 50% was measured for at least 1 month. A continuous decolorization experiment was set up by a packed-bed reactor based on colonized apple residues achieving a performance of 100 mg dye L⁻¹ day⁻¹ at a retention time of 50 h.     

Immobilization of laccase from the white rot fungus Coriolopsis polyzona and use of the immobilized biocatalyst for the continuous elimination of endocrine disrupting chemicals

Laccase from the white rot fungus strain Coriolopsis polyzona was immobilized covalently on the diatomaceous earth support Celite^® R-633 using different strategies. A first methodology involved the sequential activation of the support surface with γ-aminopropyltriethoxysilane followed by the reaction of the functionalized surface with glutaraldehyde (GLU) or glyoxal (GLY) and the immobilization of laccase on the activated surface. Another strategy tested the simultaneous internal cross-linking of the protein with GLU or GLY and the immobilization of the laccase on the silanized surface. Finally, these two strategies were modified to test the impact of the concomitant addition of bovine serum albumin (BSA) as a stabilizing agent during the immobilization steps. The highest laccase activity and the greatest degree of activity recovery (tested using 2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as the substrate) were achieved by the sequential immobilization procedure using GLU as the cross-linking agent. The solid catalysts featuring internal cross-linking of the protein showed significantly higher stability against several denaturants. The Michaelis–Menten kinetic parameters with respect to ABTS revealed a higher affinity for this substrate in the case of the sequential procedure compared to the simultaneous approach. The biocatalyst formed using GLU in the sequential procedure was applied in a packed bed reactor for the continuous treatment of 5 mg l⁻¹ solutions of the endocrine disrupting chemicals (EDCs) nonylphenol (NP), bisphenol A (BPA) and triclosan (TCS) through repeated batch treatments. All of these EDCs could be eliminated at a contact time of less than 200 min by using, respectively, 3.75 units (U) of laccase activity for BPA and TCS and 1.88 U for NP. These performances of elimination were maintained over five consecutive treatment cycles using the same biocatalyst. This system could also remove these EDCs from 100 mg l⁻¹ solutions. The Michaelis–Menten kinetic parameters with respect to these chemicals showed a decreasing affinity of the solid biocatalyst for NP, TCS and BPA in that order.     

Measurement of Phenolic Environmental Estrogens in Women with Uterine Leiomyoma

Objectives

To investigate the effect of phenolic environmental estrogens on uterine leiomyoma from the perspective of clinical epidemiology.

Methods

Urine and blood samples were collected from Han women with uterine leiomyoma and women without uterine leiomyoma, living in Nanjing, China, between September 2011 and February 2013. A total of 156 urine samples and 214 blood samples were collected from the uterine leiomyoma group and 106 urine samples and 126 blood plasma samples from the control group. Bisphenol A (BPA), nonylphenol (NP) and octylphenol (OP) concentrations were determined by solid-phase extraction (SPE) coupled with liquid chromatography-tandem mass spectrometry (HPLC-MS/MS).

Results

Phenolic environmental estrogens in the uterine leiomyoma and control groups were compared based on: gravida>3 and gravida ≤ 3. In participants with gravida>3, urine OP concentration was significantly (P<0.05) higher in the uterine leiomyoma group than in the control group. In participants with gravida ≤ 3, urine NP concentration was significantly (P<0.05) higher in the uterine leiomyoma group compared to controls. Despite obstetric history, urine BPA mean exposure concentration was significantly (P<0.05) different between uterine leiomyoma group and control group. The urine BPA concentration was not significantly (P>0.05) different between gravida>3 and gravida ≤ 3 patients. There was no significant (P>0.05) difference in plasma concentrations of BPA, OP and NP between the leiomyoma group and control group. Mean exposure concentration and range of distribution of BPA, OP and NP plasma concentration differed between the uterine leiomyoma and control group.

Conclusion

Exposure level of phenolic environmental estrogens in human was related with leiomyoma tumorigenesis.     

Oestrogenic effects of ICI 182,780, a putative anti-oestrogen,on the secretion of oxytocin and prostaglandin F2α during oestrous cycle in the intact ewe

The effect of ICI 182,780, oestrogen antagonist, on the concentrations of oxytocin and uterine PGF_2α was investigated in intact Border Leicester Merino cross ewes during the late oestrous cycle. Twelve cyclic ewes (n=6 per group) were randomly assigned to receive, at 6 h intervals, intra-muscular injection of either peanut oil or ICI 182,780 (1.5 mg kg⁻¹ day⁻¹) in oil for 2 days, starting at 1900 h on day 13 until 1300 h on day 15 post-oestrus. Hourly blood samples were collected via a jugular catheter from 0800 h on day 14 for 37 h and then daily over days 16, 17 and 18 post-oestrus. Peripheral plasma concentrations of oxytocin, the metabolite of prostaglandin F_2α, 15-keto-13,14-dihydro-prostaglandin F_2α, (PGFM) and progesterone were measured by radioimmunoassay. All ewes treated with ICI 182,780 exhibited functional luteal regression as indicated by a marked reduction in plasma progesterone concentrations to less than 1000 pg/ml over the period of 18–36 h during sampling period on days 14 and 15 of the oestrous cycle. In five of six vehicle-treated ewes, progesterone concentrations declined between day 16 and day 18 post-oestrus. In the remaining control ewe, progesterone concentrations reach less than 1000 pg/ml within 36 h of the commencement of the sampling period. During the frequent sampling period, the number of oxytocin pulses in the ICI 182,780 treated ewes was significantly higher compared to control ewes (2.7±0.3 vs. 0.8±0.3). The mean amplitude of oxytocin pulses observed was also greater (70.4±19.5 pg/ml) in ewes treated with ICI 182,780, but was not significantly different from control ewes (33.5±12.9 pg/ml). Oxytocin pulses may however have occurred following the initial two ICI 182,780 injections but before commencing blood sampling. The oxytocin pulses were detected at a mean of 3.2±0.2 h following each injection with ICI 182,780 during blood sampling. In the ICI 182,780-treated ewes, the pulsatile pattern of plasma PGFM in jugular blood samples over the 37 h sampling period on days 14 and 15 post-oestrus had a higher amplitude (512.9±158.9 vs. 121.7±78.7 pg/ml) and pulse area (618.1±183.3 vs. 151.5±102.9 (pg/ml)τ) compared to the vehicle-treated ewes (P<0.05) respectively. The average number of PGFM pulses observed per ewe was 3.0±0.7 in the ICI 182,780-treated group and was significantly (P<0.02) higher than the number of pulses (0.5±0.3) observed in ewes treated with vehicle alone. The PGFM pulses were detected at 4.2±0.6 h following each injection with ICI 182,780 during blood sampling. The percentage of PGFM pulses that occurred coincidently with a significant elevation of oxytocin concentrations was 44.4% in ICI 182,780-treated compared to 66.6% in control ewes. We conclude that administration of oestrogen antagonist ICI 182,780 accelerated development of the luteolytic mechanism by enhancing pulsatile secretion of oxytocin and PGFM which suggests that ICI 182,780 acts as an agonist for oxytocin and prostaglandin F_2α release in intact ewes when administered at 1.5 mg/kg/day over Day 13 to 15 post-oestrus.     

Substrate recognition and hydrolysis by a fungal xyloglucan-specific family 12 hydrolase

Biochemical studies to elucidate the structural basis for xyloglucan specificity among GH12 xyloglucanases are lacking. Accordingly, the substrate specificity of a GH12 xyloglucanase from Aspergillus niger (AnXEG12A) was investigated using pea xyloglucan and 12 xylogluco-oligosaccharides, and data were compared to a structural model of the enzyme. The specific activity of AnXEG12A with pea xyloglucan was 113 μmol min⁻¹ mg⁻¹, and apparent k_cat and K_m values were 49 s⁻¹ and 0.54 mg mL⁻¹, respectively. These values are similar to previously published results using xyloglucan from tamarind seed, and suggest that substrate fucosylation does not affect the specific activity of this enzyme. AnXEG12A preferred xylogluco-oligosaccharides containing more than six glucose units, and with xylose substitution at the −3 and +1 subsites. The specific activities of AnXEG12A on 100 μM XXXGXXXG and 100 μM XLLGXLLG were 60 ± 4 and 72 ± 9 μmol min⁻¹ mg⁻¹, respectively. AnXEG12A did not hydrolyze XXXXXXXG, consistent with other data that demonstrate the requirement for an unbranched glucose residue for hydrolysis by this enzyme.     

Transferrin receptor-1 iron-acquisition pathway — Synthesis,kinetics, thermodynamics and rapid cellular internalization of a holotransferrin–maghemite nanoparticle construct

Background

Targeting nanoobjects via the iron-acquisition pathway is always reported slower than the transferrin/receptor endocytosis. Is there a remedy?

Methods

Maghemite superparamagnetic and theragnostic nanoparticles (diameter 8.6 nm) were synthesized, coated with 3-aminopropyltriethoxysilane (NP) and coupled to four holotransferrin (TFe₂) by amide bonds (TFe₂–NP). The constructs were characterized by X-ray diffraction, transmission electron microscopy, FTIR, X-ray Electron Spectroscopy, Inductively Coupled Plasma with Atomic Emission Spectrometry. The in-vitro protein/protein interaction of TFe₂–NP with transferrin receptor-1 (R1) and endocytosis in HeLa cells were investigated spectrophotometrically, by fast T-jump kinetics and confocal microscopy.

Results

In-vitro, R1 interacts with TFe₂–NP with an overall dissociation constant K_D = 11 nM. This interaction occurs in two steps: in the first, the C-lobe of the TFe₂–NP interacts with R1 in 50 μs: second-order rate constant, k₁ = 6 × 10¹⁰ M^− 1 s^− 1; first-order rate constant, k_− 1 = 9 × 10⁴ s^− 1; dissociation constant, K_1d = 1.5 μM. In the second step, the protein/protein adduct undergoes a slow (10,000 s) change in conformation to reach equilibrium. This mechanism is identical to that occurring with the free TFe₂. In HeLa cells, TFe₂–NP is internalized in the cytosol in less than 15 min.

Conclusion

This is the first time that a nanoparticle–transferrin construct is shown to interact with R1 and is internalized in time scales similar to those of the free holotransferrin.

General significance

TFe₂–NP behaves as free TFe₂ and constitutes a model for rapidly targeting theragnostic devices via the main iron-acquisition pathway.     

Genetic association between IL-17F gene polymorphisms and the pathogenesis of Graves' Disease in the Han Chinese population

Background

Graves' Disease (GD) is a common and complex disorder, with a strong hereditary component. IL-17F is a potent cytokine and a potential contributor to the etiology of various human autoimmune diseases. In the present study, we focused on the relationship between polymorphisms in the IL-17F gene and GD susceptibility through a case–control association study in two independent Chinese cohorts.

Methods

Our pilot study was performed on a cohort from Shanghai, which included 757 GD patients and 741 healthy controls. Our replication cohort was from Xiamen, consisting of 434 GD patients and 420 healthy controls. We selected four tag SNPs (rs763780, rs2397084, rs9463772 and rs761167) within the IL-17F gene to conduct a genotyping analysis.

Results

In the Shanghai cohort, the rs9463772 polymorphism showed a significant association with GD and Graves' Disease-associated Ophthalmopathy (GO) patients (P_allele = 7 × 10^− 5 and 7.4 × 10^− 3 for GD and GO patients, respectively). The rs763780 polymorphism was found to have only a difference in genotype distribution between GD individuals and healthy controls (P = 0.017). In the replication study, we confirmed the association between the rs9463772 polymorphism and GD susceptibility. Haplotype analysis showed that the haplotype of the four SNPs (GCTT) was associated with a significant risk of GD in the Shanghai cohort (P = 7.9 × 10^− 3).

Conclusion

Our results suggest that polymorphisms in the IL-17F gene increase the risk of Graves' Disease and that IL-17F is therefore a good candidate gene for Graves' Disease prediction in the Han Chinese population.     

The effects of bee (Apis mellifera) venom phospholipase A2 on Trypanosoma brucei brucei and enterobacteria

The potential role of phospholipases in trypanosomiasis was investigated using bee venom phospholipase A2 (bvPLA2) as a model. The effects of bvPLA2 on the survival of Trypanosoma brucei brucei, 2 h and 12 h cultures of Enterobacter cloacae, Escherichia coli, Citrobacter freundii were studied. About 1 mg ml⁻¹ bvPLA2 was trypanocidal after 30 min. Some growth occurred at lower concentrations up to 2 h after treatment but viability decreased up to 8 h. Even very low concentrations of bvPLA2 (10⁻¹² mg ml⁻¹) had some trypanocidal activity. Bee venom PLA2 was bactericidal to 2 h bacterial cultures but bacteriostatic to 12 h ones. Minimum bactericidal concentrations were 10⁻⁵-10⁻⁶ mg ml⁻¹. The results showed that bvPLA2 had significant trypanocidal and antibacterial effects on Gram-negative bacteria. The relationship to events occurring during infection is discussed. Phospholipases may play a role in increased endotoxin levels in trypanosomiasis.     

Community metabolism and buffering capacity of nitrogen in a ruppia cirrhosa meadow

Fluxes of oxygen, inorganic nitrogen (DIN) and denitrification (isotope pairing) were measured from January 1997 to February 1998 via intact cores incubation in a shallow brackish area within the eutrophic Valli di Comacchio (northern Adriatic coast, Italy). Rates were measured in the light and in the dark in sediments colonized by the rooted macrophyte Ruppia cirrhosa and in adjacent sediments with benthic microalgae. Ruppia biomass (25-414 g DW m^− 2) exhibited a seasonal evolution whilst that of microphytobenthos (12-66 mg chl a m^− 2) was more erratic. Net (NP) and gross (GP) primary productivity was 1.15 and 6.89 mol C m^− 2y^− 1 for bare and 25.4 and 51.7 mol C m^− 2y^− 1 for Ruppia vegetated sediments. Nitrogen pools in Ruppia standing stock varied from 43.6 to 631.4 (annual average 201.2) mmol N m^− 2; the macrophyte N content was correlated with DIN concentration in the water column. Estimated N pool in microphytobenthos was one order of magnitude lower (from 2.4 to 14.5 mmol N m^− 2, annual average 7.2). Theoretical DIN assimilation calculated from NP was 127.8 and 1112.6 mmol N m^− 2y^− 1 whilst that calculated from GP was 765 and 2282 mmol N m^− 2y^− 1 for microphytobenthos and Ruppia respectively. Measured annual fluxes of DIN were 974.6 and − 577 mmol N m^− 2y^− 1 in bare and Ruppia vegetated sediments meaning that the two sites were a source and sink for DIN and that from 25 to 50% of Ruppia annual DIN requirements came from the water column. During the period of this study total denitrification was lower in the macrophyte colonized (92.3 mmol N m^− 2y^− 1) compared to bare sediments (163.3 mmol N m^− 2y^− 1) as a probable consequence of higher competition between denitrifiers and phanerogams. At both sites the ratio between denitrification of water column nitrate (D_W) and denitrification coupled to nitrification (D_N) was >1.6 due to little oxygen penetration in reducing sediments (< 1.2 mm) and scarce nitrification activity. D_W (0-35 µmol N m^− 2h^− 1) was significantly correlated with water column NO₃⁻  (2-16 µM). Theoretical DIN assimilation to denitrification ratio varied from 12.0 to 24.8 for Ruppia vegetated and from 0.8 to 4.7 for unvegetated sediments.At Valle Smarlacca, Ruppia may influence nitrogen cycling by incorporating large DIN pools in biomass which is scattered in surrounding areas and fuels intense bacterial activity. With increasing anthropogenic nutrient input and insignificant organic matter export in the open sea the already severe eutrophic conditions are enhanced and may accelerate the decline of the macrophyte meadow.     

Influence of exogenous salicylic acid on flavonolignans and lipoxygenase activity in the hairy root cultures of Silybum marianum

Silymarin is one of the most potent antioxidant so far developed from plant sources used as hepatoprotectants. Influence of different concentrations (0, 1, 2, 4, 6 and 8 mg/50 ml culture) and exposure time (24, 48, 72, 96 and 120 h) of salicylic acid on lipoxygenase activity, linoleic acid content, growth and production of silymarin in hairy root cultures of S. marianum were investigated. Detection and identification of flavonolignans was carried out by high performance liquid chromatograph method. Salicylic acid enhanced silymarin production (1.89 mg g⁻¹ DW). The optimal feeding condition was the addition of salicylic acid (6 mg/50 ml culture) after 24 h in which the silymarin content was 2.42 times higher than the control (0.78 mg g⁻¹ DW). The content of silybin, isosilybin, silychristin, silydianin and taxifolin were 0.703, 0.017, 0.289, 0.02 and 0.863 mg g⁻¹ DW respectively in these samples, while in non-treated hairy roots were 0.027, 0.046, 0.23, 0.022 and 0.453 respectively. Lipoxygenase activity also affected by elicitation. lipoxygenase activity increased 24 h after treatment by ∼1.57- fold (0.21 Δ OD₂₃₄/mg protein min⁻¹). Upon elicitation with salicylic acid, linoleic acid content of hairy roots (38.26 mg g⁻¹ DW) were also elevated after 24 h, in which the linoleic acid content was 2.37 times higher than the control (16.1 mg g⁻¹ DW). It is feasible that elicitation with salicylic acid regulates the jasmonate pathway, which in turn mediates the elicitor-induced accumulation of silymarin.     

The effects of photoperiod and light intensity on the sporulation of Brazilian and Norwegian isolates of Neozygites floridana

The objective of this study was to determine the effects of light intensity and duration (photoperiod) on the sporulation (discharge of primary conidia) and conidia germination (from non-infective primary conidia to infective capilliconidia) of Neozygites floridana isolates from Tetranychus urticae originating from Norway and Brazil. Two light intensities (40 and 208 μmol m⁻² s⁻¹), three photoperiods (24 h of continuous light (24 h D), 12 h of darkness followed by 12 h of light (12 h D: 12 h L) and 24 h of continuous darkness (24 h D)) and two temperatures (18 °C and 23 °C) were tested. The fungus produced similar amounts of primary conidia and capilliconidia at 12 h D:12 h and 24 h D, indicating that the fungus discharges almost all of its conidia during the first 12 h of darkness. Light had less of an effect on the production of primary conidia than on capilliconidia formation. At 24 h L, capilliconidia formation was significantly lower for all tested light intensities, temperatures and isolates compared to 12 h D:12 h L and 24 h D. At both light intensities, 24 h L resulted in a significantly lower capilliconidia formation for the Norwegian isolate compared to the Brazilian isolate. Our data suggest that, even though 24 h L reduced sporulation, some capilliconidia formation may occur at the low light intensities found on the underside of strawberry leaves during parts of the day as well as the top of a non-shaded strawberry leaf during the dim evening and morning hours in the tropics and during the dim, long summer days in temperate regions.     

Macrophage ABCA5 deficiency influences cellular cholesterol efflux and increases susceptibility to atherosclerosis in female LDLr knockout mice

Objectives

To determine the role of macrophage ATP-binding cassette transporter A5 (ABCA5) in cellular cholesterol homeostasis and atherosclerotic lesion development.

Methods and results

Chimeras with dysfunctional macrophage ABCA5 (ABCA5^−M/−M) were generated by transplantation of bone marrow from ABCA5 knockout (ABCA5^−/−) mice into irradiated LDLr^−/− mice. In vitro, bone marrow-derived macrophages from ABCA5^−M/−M chimeras exhibited a 29% (P < 0.001) decrease in cholesterol efflux to HDL, whereas a 21% (P = 0.07) increase in cholesterol efflux to apoA-I was observed. Interestingly, expression of ABCA1, but not ABCG1, was up-regulated in absence of functional ABCA5 in macrophages. To induce atherosclerosis, the transplanted LDLr^−/− mice were fed a high-cholesterol Western-type diet (WTD) for 6, 10, or 18 weeks, allowing analysis of effects on initial as well as advanced lesion development. Atherosclerosis development was not affected in male ABCA5^−M/−M chimeras after 6, 10, and 18 weeks WTD feeding. However, female ABCA5^−M/−M chimeras did develop significantly (P < 0.05) larger aortic root lesions as compared with female controls after 6 and 10 weeks WTD feeding.

Conclusions

ABCA5 influences macrophage cholesterol efflux, and selective disruption of ABCA5 in macrophages leads to increased atherosclerotic lesion development in female LDLr^−/− mice.     

Biochemical characterization of a novel cycloisomaltooligosaccharide glucanotransferase from Paenibacillus sp. 598K

Cycloisomaltooligosaccharide glucanotransferase (CITase; EC 2.4.1.248), a member of the glycoside hydrolase family 66 (GH66), catalyzes the intramolecular transglucosylation of dextran to produce cycloisomaltooligosaccharides (CIs; cyclodextrans) of varying lengths. Eight CI-producing bacteria have been found; however, CITase from Bacillus circulans T-3040 (CITase-T3040) is the only CI-producing enzyme that has been characterized to date. In this study, we report the gene cloning, enzyme characterization, and analysis of essential Asp and Glu residues of a novel CITase from Paenibacillus sp. 598K (CITase-598K). The cit genes from T-3040 and 598K strains were expressed recombinantly, and the properties of Escherichia coli recombinant enzymes were compared. The two CITases exhibited high primary amino acid sequence identity (67%). The major product of CITase-598K was cycloisomaltoheptaose (CI-7), whereas that of CITase-T3040 was cycloisomaltooctaose (CI-8). Some of the properties of CITase-598K are more favorable for practical use compared with CITase-T3040, i.e., the thermal stability for CITase-598K (≤ 50 °C) was 10 °C higher than that for CITase-T3040 (≤ 40 °C); the k_cat/K_M value of CITase-598K was approximately two times higher (32.2 s^− 1 mM^− 1) than that of CITase-T3040 (17.8 s^− 1 mM^− 1). Isomaltotetraose was the smallest substrate for both CITases. When isomaltoheptaose or smaller substrates were used, a lag time was observed before the intramolecular transglucosylation reaction began. As substrate length increased, the lag time shortened. Catalytically important residues of CITase-598K were predicted to be Asp144, Asp269, and Glu341. These findings will serve as a basis for understanding the reaction mechanism and substrate recognition of GH66 enzymes.     

Increased renal semicarbazide-sensitive amine oxidase activity and methylglyoxal levels in aristolochic acid-induced nephrotoxicity

Aims

Aristolochic acid (AA) nephrotoxicity is related to accumulation of methylglyoxal (MGO) and N^ε-(carboxymethyl)lysine (CML) in the mouse kidney. We studied the activity of renal semicarbazide-sensitive amine oxidase (SSAO), a key enzyme involved in MGO generation, in AA-treated mice, and investigated nephroprotective effects produced by metformin, a MGO scavenger.

Methods

Mice were orally administered water or metformin for 15 days (12 or 24 mg kg^− 1 day^− 1), and injected AA (5 mg kg^− 1 day^− 1) intraperitoneally for 8 days starting on day 8. Renal function was studied, and histopathological examination, determination of renal SSAO activity, and measurement of MGO levels were performed.

Key findings

Compared to control mice, AA-injected mice showed significant renal damage and approximately 2.7-fold greater renal SSAO activity (p < 0.05). Further, compared to control treatment, administration of 12 mg/kg metformin inhibited formation of renal lesions, and significantly decreased renal MGO levels (37.33 ± 9.78 vs. 5.89 ± 2.64 μg/mg of protein, respectively, p < 0.01). In the AA-treated mice, metformin also inhibited the accumulation of CML in renal tubules, but did not affect SSAO activity.

Significance

This study is the first to show elevated renal SSAO activity in AA-treated mice, which could be involved in MGO accumulation. Moreover, MGO scavenging by metformin reduces AA nephrotoxicity. These findings suggest that reducing MGO accumulation produces nephroprotection, revealing new therapeutic strategies for the management. SSAO is a key enzyme involved in MGO generation, and consequently, inhibition of renal SSAO activity is worth investigating in AA nephrotoxicity and other renal pathologies further.     

D-Lys-GHRP-6 antagonizes the effect of unacylated but not of acylated ghrelin on the growth of HECa10 murine endothelial cells

Recent studies demonstrate that ghrelin can be an endogenous regulator of angiogenesis. We studied direct effects of human acylated (hAG) and unacylated (hUAG) ghrelin, as well as of rat acylated ghrelin (rAG) on the growth of HECa10 murine endothelial cells. Ghrelin was applied separately or together with D-Lys³-GHRP-6, which is commonly used as an antagonist of ghrelin receptor type 1a – GHS-R1a. The growth of HECa10 cells was assessed with Mosmann and in selected study conditions also with BrdU and TUNEL methods. Both hAG and hUAG (10⁻⁵ M to 10⁻¹² M) inhibited the growth of HECa10 cells in 24 h and 72 h cultures. Similarly, rAG decreased the growth of the cells after 24 h (10⁻⁷ M and 10⁻¹¹ M), and after 72 h (10⁻⁷ M, 10⁻⁸ M and 10⁻¹¹ M). Unexpectedly, D-Lys³-GHRP-6 itself also inhibited the growth of these cells at 10⁻⁴ to 10⁻⁶ M in 24 h, 48 h (dose–response effect) and 72 h cultures. D-Lys³-GHRP-6 did not modify the inhibitory effect of rAG. However, D-Lys³-GHRP-6 at the concentration of 10⁻⁴ M diminished, abolished or even reversed the inhibitory effect of hUAG in 72 h culture and this was dependent on ghrelin concentrations. These data indicate that both AG and UAG have antiangiogenic properties at least at the level of endothelial growth, through decreased metabolic activity of the cells or stimulation of apoptosis. D-Lys³-GHRP-6 (inhibitor of GHS-R1a) seems not to be an appropriate antagonist in this experimental condition. Similar effects of these substances on HECa10 cells suggest that they are not mediated by GHS-R1a.