Protective role of zinc in liver damage in experimental diabetes demonstrated via different biochemical parameters

Diabetes mellitus is a serious worldwide metabolic disease, which is accompanied by hyperglycaemia and affects all organs and body system. Zinc (Zn) is a basic cofactor for many enzymes, which also plays an important role in stabilising the structure of insulin. Liver is the most important target organ after pancreas in diabetic complications. In this study, we aimed to investigate the protective role of Zn in liver damage in streptozotocin (STZ)‐induced diabetes mellitus. There are four experimental groups of female Swiss albino rats: group I: control; group II: control + ZnSO₄; group III: STZ‐induced diabetic animals and group IV: STZ‐diabetic + ZnSO₄. To induce diabetes, STZ was injected intraperitoneally (65 mg/kg). ZnSO₄ (100 mg/kg) was given daily to groups II and IV by gavage for 60 days. At the end of the experiment, rats were killed under anaesthesia and liver tissues were collected. In the diabetic group, hexose, hexosamine, fucose, sialic acid levels, arginase, adenosine deaminase, tissue factor activities and protein carbonyl levels increased, whereas catalase, superoxide dismutase, glutathione‐S‐transferase, glutathione peroxidase, glutathione reductase and Na⁺/K⁺‐ATPase activities decreased. The administration of Zn to the diabetic group reversed all the negative effects/activities. According to these results, we can suggest that Zn has a protective role against STZ‐induced diabetic liver damage.     

Enhanced dewatering of waste-activated sludge by composite hydrolysis enzymes

The feasibility of composite hydrolysis enzymes in enhanced dewatering of waste-activated sludge (WAS) was verified in this study. A Pearson correlation analysis was conducted to explore the roles of different extracellular polymeric substance (EPS) fractions on WAS dewaterability. The results indicated that tightly bound EPS (TB-EPS) was released into the liquid phase consistently during enzymatic hydrolysis to form soluble EPS (S-EPS) and loosely bound EPS and that the TB-EPS content was positively correlated with the capillary suction time of WAS. A kinetic analysis was carried out to gain further insights into the kinetic variation in TB-EPS removal. It was found that TB-EPS reduction fit a first-order kinetic model and that mild temperature (25–30 °C) and a slightly acidic condition were favorable for the improvement of enzyme activity. Solid phase extraction combined with UV–Vis spectroscopy analysis was used to characterize the processes of migration and transformation of the hydrophobic (HPO), transphilic and hydrophilic (HPI) fractions in EPS during the enzymatic process. The results revealed that HPO and HPI were mainly composed of PN and PS, respectively, and that the enzymatic hydrolysis could enhance the transformation of HPI from TB-EPS to S-EPS, which was the dominant mechanism of improving WAS dewaterability.     

Knockdown of TRPV4 suppresses osteoclast differentiation and osteoporosis by inhibiting autophagy through Ca2+–calcineurin–NFATc1 pathway

The aim of this study is to evaluate the effect of transient receptor potential vanilloid 4 (TRPV4) on osteoclast differentiation and osteoporosis, and to investigate the underlying mechanism. The results showed that TRPV4 expression and intracellular Ca²⁺ concentration were significantly upregulated in macrophage colony-stimulating factor (M-CSF)-stimulated and receptor activator of nuclear factor κΒ ligand (RANKL)-stimulated RAW264.7 cells. Furthermore, TRPV4 overexpression further increased the M-CSF- and RANKL-induced number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts and expression of osteoclastogenesis-related genes (TRAP, c-Fos, and nuclear factor of activated T cells [NFATc1]), activated the Ca ²⁺–calcineurin–NFATc1 signaling and increased autophagy-related proteins (light chain [LC] 3II and Beclin-1) during osteoclast differentiation. In contrast, TRPV4 knockdown exerted the opposite effects. Mechanically, inhibition of Ca ²⁺–calcineurin–NFATc1 signaling by FK506 or 11R-VIVIT abrogated the TRPV4 overexpression-induced osteoclast differentiation and autophagy induction. Moreover, suppression of autophagy by 3-methyladenine attenuated the TRPV4-induced osteoclast differentiation. In addition, short hairpin RNA TRPV4-lentivirus administration significantly diminished the increased levels of several osteoclastogenesis-related genes (RANKL, TRAP, and tumor necrosis factor-α), alleviated the disturbed microarchitecture of lumbar vertebrae, restored the decreased bone mineral density, ratio of bone volume to total tissue volume, trabecular thickness, and trabecular number, and diminished the increased trabecular separation, in ovariectomy (OVX)-induced osteoporosis mice. Consistent with the in vitro data, TRPV4 knockdown significantly decreased the induced number of TRAP-positive osteoclasts, the increased LC3 and NFATc1 expression in the lumbar vertebrae of OVX mice. In conclusion, TRPV4 knockdown suppresses osteoclast differentiation and osteoporosis by inhibiting autophagy through Ca ²⁺–calcineurin–NFATc1 pathway.     

Interaction of the corepressor Alien with DAX-1 is abrogated by mutations of DAX-1 involved in adrenal hypoplasia congenita

DAX-1 is an unusual member of the nuclear hormone receptor (NHR) superfamily. Lack of DAX-1-mediated silencing leads to adrenal hypoplasia congenita and hypogonadotropic hypogonadism. Gene silencing through NHRs such as the thyroid hormone receptor (TR) is mediated by corepressors. We have previously characterized a novel corepressor, termed Alien, which interacts with TR and the ecdysone receptor but not with the retinoic acid receptors RAR or RXR. Here, we show that DAX-1 interacts with the corepressor Alien but not with the corepressor SMRT. This interaction is mediated by the DAX-1-silencing domain. Naturally occurring mutants of the DAX-1 gene fail to interact with Alien and have lost silencing function. Because the silencing domain of DAX-1 is unusual for NHRs, we mapped the interaction of Alien with DAX-1 and with TR. We show that Alien exhibits different binding characteristics to DAX-1 and TR. Furthermore, Northern experiments demonstrate that Alien is expressed in the adrenal gland and testis in tissues where DAX-1 is specifically expressed. Interestingly, a novel adrenal gland-specific mRNA of Alien was discovered. Thus, the impairment of Alien binding seems to play an important role in the pathogenesis mediated by DAX-1 mutants.     

Partial purification and characterization of the organic solvent-tolerant lipase produced by Pseudomonas fluorescens RB02-3 isolated from milk

Eighty-five putative Pseudomonas isolates were obtained from various raw milk and pasteurized milk samples using Pseudomonas CFC agar. Among them, 36 isolates were identified as Pseudomonas fluorescens, and one isolate was identified as Pseudomonas putida. Lipase activity of the strains was quantitatively measured by the spectrophotometric method using p-nitrophenyl palmitate (p-NPP) as substrate. Detected lipase activity of the strains was between 10.03 U/mL and 22.16 U/mL. Pseudomonas fluorescens RB02-3 possessed the highest lipase activity. The extracellular lipase of P. fluorescens RB02-3 strain was homogeneously purified using a combination of ammonium sulfate precipitation, dialysis, and gel filtration column chromatography. This purification procedure resulted in 2.97-fold purification with 20.3% recovery. The enzyme was characterized, and exhibited maximum activity at pH 7.0 and 50 °C; after it was incubated for 1 h it was activated in the presence of hexane, ethyl acetate, isopropanol, and ethanol and remained stable after the incubation was extended for 2 hr. The lipase was slightly inhibited in the presence of Zn2+, Co2+, Cu2+, Ni2+ salts, and ethylenediamine tetraacetic acid (EDTA), whereas Cd2+, sodium dodecyl sulfate (SDS), and Tween-80 had no effect on its activity.     

Effect of Nitric Oxide on Anammox Bacteria

The effects of nitrogen oxides on anammox bacteria are not well known. Therefore, anammox bacteria were exposed to 3,500 ppm nitric oxide (NO) in the gas phase. The anammox bacteria were not inhibited by the high NO concentration but rather used it to oxidize additional ammonium to dinitrogen gas under conditions relevant to wastewater treatment.Nitric oxide (NO) has several different roles in bacteria, fungi, and mammals (24). In nitrogen cycle bacteria, it acts as an intermediate and cell communication/signal transduction molecule. On the other hand, NO is a highly reactive and toxic compound that contributes to ozone depletion and air pollution (5). Due to its reactive nature, many bacteria employ an arsenal of proteins (those encoded by norVW, as well as bacterial globins, heme proteins, etc.) that are used to detoxify NO to the less-reactive and more-stable nitrous oxide (N₂O) (24). Still, N₂O is a very effective greenhouse gas and an unfavorable constituent in the off-gases from nitrification/denitrification nitrogen removal systems (4). The presence of gene(s) encoding cytochrome cd₁ nitrite reductase (EMBL accession no. {"type":"entrez-protein","attrs":{"text":"CAJ74898","term_id":"91201838","term_text":"CAJ74898"}}CAJ74898), flavorubredoxin NorVW (accession no. {"type":"entrez-protein","attrs":{"text":"CAJ73918","term_id":"91200863","term_text":"CAJ73918"}}CAJ73918 and {"type":"entrez-protein","attrs":{"text":"CAJ73688","term_id":"91200638","term_text":"CAJ73688"}}CAJ73688), and bacterial hemoglobin (accession no. {"type":"entrez-protein","attrs":{"text":"CAJ72702","term_id":"91203063","term_text":"CAJ72702"}}CAJ72702) in the genome of Kuenenia stuttgartiensis led to the proposal that NO also plays this dual role (metabolic versus toxic) in anammox bacteria (Fig. ​(Fig.1)1) (10, 20). This has ramifications for both application and metabolism of anammox bacteria. The source of NO in an anammox reactor could be the activity of other community members (ammonium-oxidizing or denitrifying bacteria) or high concentrations of nitrite in the influent wastewater stream. Full-scale anammox reactors typically contain a significant population of ammonium-oxidizing bacteria (AOB). In the single nitritation-anammox reactors, these carry out the conversion of 50% of the ammonium in the wastewater to nitrite (6). It has been shown that AOB may produce significant amounts of NO (2, 7), and recently it was reported that NO and N₂O could be emitted from these reactors up to 0.005 and 1.2% of the total nitrogen load to the reactor, respectively (6, 23). NO may inhibit the anammox bacteria and could also be further reduced to N₂O in these reactors (6, 23). It is presently unknown whether anammox bacteria contribute to the NO or N₂O emissions, although it has been suggested previously that anammox bacteria do not produce N₂O under physiologically relevant conditions (10). Nevertheless, if conversion of NO could be coupled to anaerobic ammonium oxidation, the toxic air pollutant NO would facilitate further removal of ammonium in full-scale anammox bioreactors. In the present study, we investigated the effect of very high NO fluxes on anammox bacteria.Open in a separate windowFIG. 1.The hypothetical anammox pathway with possible routes of NO removal. Solid black arrows: anammox pathway, including nitrite oxidation to nitrate; gray arrow, possible detoxification pathway to N₂O (not observed in the bioreactor); dashed gray arrow, NO oxidation to nitrite/nitrate (not possible under anoxic conditions).NO has been described many times as a potent inhibitor of nitrogen cycle bacteria; aerobic ammonium oxidizers, nitrite oxidizers, and denitrifiers were all inhibited by concentrations as low as a few micromolar units (1, 18, 24). In a previous study, it was suggested that “Candidatus Brocadia anammoxidans” could tolerate up to 600 ppm NO (approximately 1 mg NO·day⁻¹ NO load) (16). In the reported experiments, without direct measurement of nitrous oxide (N₂O) in the effluent gas stream, it was postulated that NO was reduced to N₂O (16). In the present study, we used a carefully monitored sequencing batch reactor (SBR) to further our understanding of the effect and fate of NO in a laboratory-scale anammox reactor under conditions which are relevant in wastewater treatment plants.An SBR (working volume, 3.5 liters) consisting of approximately 80% of the anammox bacterium “Candidatus Brocadia fulgida” and no detectable aerobic ammonium oxidizers (determined by fluorescence in situ hybridization (FISH) as described previously [15]) was used in the present study. Before the first introduction of NO into the reactor, the influent (synthetic wastewater) (21) was supplied to the reactor at a flow rate of 1.4 ml·min⁻¹ with nitrite and ammonium concentrations (assayed as previously described [9]) at 45 and 39 mM, respectively (corresponding to a total of 2,370 mg N·day⁻¹). All nitrite was consumed in the reactor, while 2 mM ammonium was still present in the effluent. For every 1 mol of ammonium, 1.22 mol of nitrite was consumed, similar to the previously determined anammox stoichiometry (19). NO was first introduced at a concentration of 400 to 600 ppm in the gas phase at a flow rate of 10 ml/min (CLD 700EL chemiluminescence NOx analyzer, detection limit of 0.1 ppm NO, with 15 ml/min Ar/CO₂ as the dilution gas [a load of 25 to 28 mg NO·day⁻¹]; EcoPhysics, Michigan). During this period, 45% (±6%) of the supplied NO was removed from the system. Initially, there was no detectable change in the ammonium and nitrite removal efficiencies and no detectable nitrous oxide (N₂O) in the flue gas (analyzed with an Agilent 6890 gas chromatograph). It is most likely that NO was converted to N₂, but the increase in the N₂ concentrations in the off-gas was below the detection limit (1,000 ppm).At day 49, the influent NO concentration was increased to 3,500 ppm (640 mg NO·day⁻¹ load). Simultaneously, the stirring speed of the reactor was increased from 200 to 600 rpm to enable better mass transfer to the flocculent anammox biomass. The increase in the stirring speed did not result in any disturbance in the floc size and settling ability of the biomass but did lead to a much higher level of NO removal (128 mg NO·day⁻¹) by the anammox bacteria. The converted NO could theoretically be converted to N₂O via detoxification enzymes or coupled to ammonium oxidation (Fig. ​(Fig.1).1). Surprisingly, there was no change in the nitrite removal capacity of the bioreactor, suggesting that NO was not a substrate preferred over nitrite. Nitrate concentrations (assayed according to the method in reference 9) were stable around 7.2 mM (±0.7 mM). Theoretically, as anammox bacteria reduce NO, they could oxidize a larger proportion of nitrite to nitrate (Fig. ​(Fig.1)1) to increase their capacity for CO₂ fixation; however, such an increase in nitrate production was not observed (or could not be discriminated by the method used [sensitivity, 100 μM]). During this phase of the experiment, the effluent ammonium concentration gradually decreased to below the detection limit (Fig. ​(Fig.2).2). There was only a minimal N₂O (0.6 ppm) emission from the system, and the total N₂ production increased from 3,060 to 3,680 mg N₂·day⁻¹. This indicated that NO reduction was coupled to the catabolism of the anammox bacteria rather than being detoxified by anammox or other community members. To the best of our knowledge, this was the first time that such a high load of NO was not found to be toxic to the nitrogen cycle bacteria. In a previous study, an NO load of 1 mg NO·day⁻¹ was reported to be toxic to anammox bacteria, most probably due to the fact that the experiments were conducted with biomass that had a 100-fold lower cell density and 10-fold lower activity compared to the current enrichment cultures. Furthermore, the NO conversion in the current experiments was stoichiometrically coupled to ammonium oxidation and not converted to N₂O, indicating that the previously reported N₂O emissions from full-scale anammox bioreactors originated not with the anammox bacteria but rather with other community members as hypothesized previously (8).Open in a separate windowFIG. 2.Ammonium concentration in the effluent of the anammox bioreactor. Dashed lines indicate the trend of effluent ammonium concentration during different phases of the reactor operation. Black arrows indicate the manipulations to influent NO stream, and the gray arrow points to an increase in the influent ammonium concentration. d, day.To determine if there could be more NO-dependent ammonium removal, the influent ammonium concentration was first increased to 41 mM (day 80) and then to 43 mM (day 81). This resulted in a slow but gradual increase in the effluent ammonium concentration, and additional ammonium did not appear to be completely converted, most probably due to NO mass transfer limitations. As a result of the higher level of ammonium removal, the observed anammox stoichiometry in the reactor decreased from 1.22 to 0.91 (nitrite/ammonium). Between days 95 and 131, the NO supply to the reactor was turned off, which resulted in an average ammonium concentration of 3.3 mM (±0.9 mM) in the effluent. Following this period, on day 132, the NO load on the reactor was increased back to 640 mg NO·day⁻¹ (Fig. ​(Fig.2).2). As a result, the effluent ammonium concentration gradually decreased again to an average of 1.5 mM (±0.36 mM). The highest level of NO removal achieved in this period was 371 mg NO·day⁻¹. When the NO supply was turned off on day 165, ammonium concentrations increased back to 3.5 mM (±0.71 mM).During the course of the experiment, the biodiversity of the reactor was monitored using FISH and 16S rRNA gene sequence analysis as described previously (15) with probes specific to eubacteria (3), Planctomycetes (13), anammox bacteria (15), “Ca. Brocadia fulgida” (11), and a variety of aerobic ammonium-oxidizing bacteria (12, 22). Before the experiments started and throughout the cultivation of the anammox bacteria with NO, the only detectable anammox species (with FISH and 16S rRNA gene sequence analysis) was “Candidatus Brocadia fulgida.”In the present study, we showed that 2 mM ammonium (4.5% of the influent concentration) could be removed by anammox bacteria via direct coupling to NO reduction. These observations support the proposal of NO as an intermediate of the anammox reaction and have two consequences for application of the anammox process for nitrogen removal. First, we obtained strong indications that previously reported N₂O emissions (6, 8) from full-scale anammox reactors were not generated by anammox bacteria. In our experiments, even under a very high load of NO, there was hardly any detectable N₂O in the effluent gas stream. The competition for nitrogen oxides by denitrifying and anammox bacteria needs further study but may ultimately be used to design operational conditions that would reduce or even prevent NO and N₂O emissions from full-scale nitritation-anammox reactors. Second, by implementing the results of this study, in the future the anammox process could be designed to remove NO from flue gases. Since NO is mostly emitted together with O₂, this could be achieved by the combination of anammox and aerobic ammonium-oxidizing bacteria, for example, with CANON (completely autotrophic nitrogen removal over nitrite)- or OLAND (oxygen-limited autotrophic nitrification-denitrification)-type reactor systems (14, 17).     

Histopathological changes induced by maneb and carbaryl on some tissues of rainbow trout, Oncorhynchus mykiss

Acute toxicity of the pesticides, maneb and carbaryl, to juvenile rainbow trout were evaluated under static-renewal test conditions. Actual concentrations of maneb ranged from 0.10 mg/L to 2.00 mg/L and carbaryl ranged from 0.20 mg/L to 3.90 mg/L. The concentrations of maneb that killed 50% of the rainbow trout (3.27 ± 0.9 g) within 24-h (24-h; LC₅₀), 48-h, 72-h and 96-h were 1.19 ± 0.12, 1.04 ± 0.11, 0.92 ± 0.12 and 0.81 ± 0.14 mg/L (95% confidence limits), respectively. LC₅₀ values of carbaryl for 24-h, 48-h, 72-h and 96-h were 2.52 ± 0.71, 2.16 ± 0.63, 1.71 ± 0.46 and 1.39 ± 0.15 mg/L, respectively. None of the unexposed control fish died and the first fish died 6 h after exposure to maneb (≥1.30 mg/L), and carbaryl (≥2.60 mg/L). Lamellar edema, separation of epithelium from lamellae, lamellar fusion, swelling of the epithelial cells and epithelial cell necrosis were observed on maneb and carbaryl exposed fish. Gills also had scattered areas of focal lamellar hyperplasia. Fish exposed to pesticides had inflammation and focal necrosis in liver, trunk kidney and spleen. Maneb and carbaryl had similar histopathological lesions. In order, the most affected organs were gill, trunk kidney and liver.     

Response of anaerobic ammonium-oxidizing bacteria to hydroxylamine

Anaerobic ammonium oxidation is a recent addition to the microbial nitrogen cycle, and its metabolic pathway, including the production and conversion of its intermediate hydrazine, is not well understood. Therefore, the effect of hydroxylamine addition on the hydrazine metabolism of anaerobic ammonium-oxidizing (anammox) bacteria was studied both experimentally and by mathematical modeling. It was observed that hydroxylamine was disproportionated biologically in the absence of nitrite into dinitrogen gas and ammonium. Little hydrazine accumulated during this process; however, rapid hydrazine production was observed when nearly all hydroxylamine was consumed. A mechanistic model is proposed in which hydrazine is suggested to be continuously produced from ammonium and hydroxylamine (possibly via nitric oxide) and subsequently oxidized to N(2). The electron acceptor for hydrazine oxidation is hydroxylamine, which is reduced to ammonium. A decrease in the hydroxylamine reduction rate, therefore, leads to a decrease in the hydrazine oxidation rate, resulting in the observed hydrazine accumulation. The proposed mechanism was verified by a mathematical model which could explain and predict most of the experimental data.     

附生植物石斛的株丛生长及营养繁殖特征

石斛(Dendrobium nobile Lindl.)为兰科多年生附生性草本植物,特化的假鳞茎是其营养贮藏器官,通过假鳞茎可实现克隆生长。该研究以野外调查发现的石斛株丛为研究材料,比较不同等级株丛假鳞茎合轴生长和高位腋芽的差异,分析高位株丛的定植方式,探讨石斛株丛生长及营养繁殖对附生环境的适应策略。结果显示:(1)石斛株丛的生长和扩大通过合轴生长的营养繁殖方式进行,假鳞茎基部具有2~3个储备芽,每年萌发1~2个新芽,随着生长年限的增加,形成大小不一的株丛。(2)株丛具有典型的高位腋芽营养繁殖特性,且主要形成于假鳞茎密集和老根密布的大株丛。(3)高位株丛母茎一端附着于附主树种上,在母茎软化和高位株丛的重力作用下,缩短了高位株丛与附主的距离,使其根系能够触及附主,完成高位株丛的定植。研究表明,附生植物石斛通过假鳞茎合轴生长的营养繁殖方式来增强并延续株丛寿命,高位腋芽的频发是株丛假鳞茎对拥挤等逆境的响应,高位株丛的定植依赖于母茎,这也是石斛对附生环境的一种生态适应策略。