Cholesterol metabolism, transport, and hepatic regulation in dairy cows during transition and early lactation

The transition from the nonlactating to the lactating state represents a critical period for dairy cow lipid metabolism because body reserves have to be mobilized to meet the increasing energy requirements for the initiation of milk production. The purpose of this study was to provide a comprehensive overview on cholesterol homeostasis in transition dairy cows by assessing in parallel plasma, milk, and hepatic tissue for key factors of cholesterol metabolism, transport, and regulation. Blood samples and liver biopsies were taken from 50 multiparous Holstein dairy cows in wk 3 antepartum (a.p.), wk 1 postpartum (p.p.), wk 4 p.p., and wk 14 p.p. Milk sampling was performed in wk 1, 4, and 14 p.p. Blood and milk lipid concentrations [triglycerides (TG), cholesterol, and lipoproteins], enzyme activities (phospholipid transfer protein and lecithin:cholesterol acyltransferase) were analyzed using enzymatic assays. Hepatic gene expression patterns of 3-hydroxy-3-methylglutaryl-coenzyme A (HMGC) synthase 1 (HMGCS1) and HMGC reductase (HMGCR), sterol regulatory element-binding factor (SREBF)-1 and -2, microsomal triglyceride transfer protein (MTTP), ATP-binding cassette transporter (ABC) A1 and ABCG1, liver X receptor (LXR) α and peroxisome proliferator activated receptor (PPAR) α and γ were measured using quantitative RT-PCR. Plasma TG, cholesterol, and lipoprotein concentrations decreased from wk 3 a.p. to a minimum in wk 1 p.p., and then gradually increased until wk 14 p.p. Compared with wk 4 p.p., phospholipid transfer protein activity was increased in wk 1 p.p., whereas lecithin:cholesterol acyltransferase activity was lowest at this period. Total cholesterol concentration and mass, and cholesterol concentration in the milk fat fraction decreased from wk 1 p.p. to wk 4 p.p. Both total and milk fat cholesterol concentration were decreased in wk 4 p.p. compared with wk 1 and 14 p.p. The mRNA abundance of genes involved in cholesterol synthesis (SREBF-2, HMGCS1, and HMGCR) markedly increased from wk 3 a.p. to wk 1 p.p., whereas SREBF-1 was downregulated. The expression of ABCA1 increased from wk 3 a.p. to wk 1 p.p., whereas ABCG1 was increased in wk 14 p.p. compared with other time points. In conclusion, hepatic expression of genes involved in the biosynthesis of cholesterol as well as the ABCA1 transporter were upregulated at the onset of lactation, whereas plasma concentrations of total cholesterol, phospholipids, lipoprotein-cholesterol, and TG were at a minimum. Thus, at the gene expression level, the liver seems to react to the increased demand for cholesterol after parturition. Whether the low plasma cholesterol and TG levels are due to impaired hepatic export mechanisms or reflect an enhanced transfer of these compounds into the milk to provide essential nutrients for the newborn remains to be elucidated.

Influence of Gilbert's syndrome on the formation of ethyl glucuronide.

A drinking experiment with participants suffering from Gilbert's syndrome was performed to study the possible influence of this glucuronidation disorder on the formation of ethyl glucuronide (EtG). Gilbert's syndrome is a rather common and, in most cases, asymptomatic congenital metabolic aberration with a prevalence of about 5 %. It is characterized by a reduction of the enzyme activity of the uridine diphosphate glucuronosyltransferase (UGT) isoform 1A1 up to 80 %. One of the glucuronidation products is EtG, which is formed in the organism following exposure to ethanol. EtG is used as a short-term marker for ethyl alcohol consumption to prove abstinence in various settings. After 2 days of abstinence from ethanol and giving a void urine sample, 30 study participants drank 0.1 L of sparkling wine (9 g ethanol). 3, 6, 12, and 24 h after drinking, urine samples were collected. 3 hours after drinking, an additional blood sample was taken, in which liver enzyme activities, ethanol, hematological parameters, and bilirubin were measured. EtG and ethyl sulfate (EtS), another short-term marker of ethanol consumption, were determined in the urine samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS); creatinine was measured photometrically. In all participants, EtG and EtS were detected in concentrations showing a wide range (EtG: 3 h sample 0.5-18.43 mg/L and 6 h sample 0.67-13.8 mg/L; EtS: 3 h sample 0.87-6.87 mg/L and 6 h sample 0.29-4.48 mg/L). No evidence of impaired EtG formation was found. Thus, EtG seems to be a suitable marker for ethanol consumption even in individuals with Gilbert's syndrome.

Direct and indirect effects of invasion by the alien tree Ailanthus altissima on riparian plant communities and ecosystem multifunctionality

Most existing studies addressing the effects of invasive species on biodiversity focus on species richness ignoring better indicators of biodiversity and better predictors of ecosystem functioning such as the diversity of evolutionary histories (phylodiversity). Moreover, no previous study has separated the direct effect of alien plants on multiple ecosystem functions simultaneously (multifunctionality) from those indirect ones mediated by the decrease on biodiversity caused by alien plants. We aimed to analyze direct and indirect effects, mediated or not by its effect on biodiversity, of the invasive tree Ailanthus altissima on ecosystem multifunctionality of riparian habitats under Mediterranean climate. We measured vegetation attributes (species richness and phylodiversity) and several surrogates of ecosystem functioning (understory plant biomass, soil enzyme activities, available phosphorous and organic matter) in plots infested by A. altissima and in control (non-invaded) ones. We used structural equation modelling to tease apart the direct and indirect effects of A. altissima on ecosystem multifunctionality. Our results suggest that lower plant species richness, phylodiversity and multifunctionality were associated to the presence of A. altissima. When analyzing each function separately, we found that biodiversity has the opposite effect of the alien plant on all the different functions measured, therefore reducing the strength of the effect (either positive or negative) of A. altissima on them. This is one of the few existing studies addressing the effect of invasive species on phylodiversity and also studying the effect of invasive species on multiple ecosystem functions simultaneously.

Mosaic pattern of sucrase isomaltase deficiency in two brothers

The pathophysiology of mucosal changes observed in infants with chronic protracted diarrhea is poorly understood. We report on two brothers suffering from a special form of sucrase isomaltase (SI) deficiency. The children presented with weight loss and dyspepsia after sucrose exposition. We performed an H respiration test, which showed a pathologic result in the younger brother. Analysis of the brush border enzyme activities showed low expression of lactase and SI. Immunoelectron microscopy of duodenal biopsies showed an isolated SI deficiency in a mosaic pattern [e.g., 42% (14%) crypt enterocytes and 64% (59%) villus enterocytes with decreased amounts of SI on microvilli], whereas lactase and aminopeptidase n (ApN) were present at the apical membrane of all cells in a normal range. The SI mosaic pattern of these patients shows that the enterocytes contain low amounts of SI on the apical membrane but express normal quantities of other disaccharidases. These findings suggest the existence of different clonal expressions or specific (posttranslational) mechanisms of postGolgi transportation for individual brush border enzymes. It remains unresolved whether the mosaic distribution is part of a normal maturation process or caused by a lack of an overall control mechanism in the expression of brush border hydrolases.

Interaction of sulfate assimilation with carbon and nitrogen metabolism in Lemna minor

Cysteine synthesis from sulfide andO-acetyl-L-serine (OAS) is a reaction interconnecting sulfate, nitrogen, and carbon assimilation. UsingLemna minor, we analyzed the effects of omission of CO2 from the atmosphere and simultaneous application of alternative carbon sources on adenosine 5′-phosphosulfate reductase (APR) and nitrate reductase (NR), the key enzymes of sulfate and nitrate assimilation, respectively. Incubation in air without CO2 led to severe decrease in APR and NR activities and mRNA levels, but ribulose-1,5-bisphosphate carboxylase/oxygenase was not considerably affected. Simultaneous addition of sucrose (Suc) prevented the reduction in enzyme activities, but not in mRNA levels. OAS, a known regulator of sulfate assimilation, could also attenuate the effect of missing CO2 on APR, but did not affect NR. When the plants were subjected to normal air after a 24-h pretreatment in air without CO2, APR and NR activities and mRNA levels recovered within the next 24 h. The addition of Suc and glucose in air without CO2 also recovered both enzyme activities, with OAS again influenced only APR.35SO4 2− feeding showed that treatment in air without CO2 severely inhibited sulfate uptake and the flux through sulfate assimilation. After a resupply of normal air or the addition of Suc, incorporation of 35S into proteins and glutathione greatly increased. OAS treatment resulted in high labeling of cysteine; the incorporation of 35S in proteins and glutathione was much less increased compared with treatment with normal air or Suc. These results corroborate the tight interconnection of sulfate, nitrate, and carbon assimilation.

Benzo(a)pyrene Metabolism and EROD and GST Biotransformation Activity in the Liver of Red- and White-Blooded Antarctic Fish.

Climate change and anthropogenic pollution are of increasing concern in remote areas such as Antarctica. The evolutionary adaptation of Antarctic notothenioid fish to the cold and stable Southern Ocean led to a low plasticity of their physiological functions, what may limit their capacity to deal with altered temperature regimes and pollution in the Antarctic environment. Using a biochemical approach, we aimed to assess the hepatic biotransformation capacities of Antarctic fish species by determining (i) the activities of ethoxyresorufin-O-deethylase (EROD) and glutathione-S-transferase (GST), and (ii) the metabolic clearance of benzo(a)pyrene by hepatic S9 supernatants. In addition, we determined the thermal sensitivity of the xenobiotic biotransformation enzymes. We investigated the xenobiotic metabolism of the red-blooded Gobionotothen gibberifrons and Notothenia rossii, the hemoglobin-less Chaenocephalus aceratus and Champsocephalus gunnari, and the rainbow trout Oncorhynchus mykiss as a reference. Our results revealed similar metabolic enzyme activities and metabolic clearance rates between red- and white-blooded Antarctic fish, but significantly lower rates in comparison to rainbow trout. Therefore, bioaccumulation factors for metabolizable lipophilic contaminants may be higher in Antarctic than in temperate fish. Likewise, the thermal adaptive capacities and flexibilities of the EROD and GST activities in Antarctic fish were significantly lower than in rainbow trout. As a consequence, increasing water temperatures in the Southern Ocean will additionally compromise the already low detoxification capacities of Antarctic fish.

Lipid, carbon and nitrogen content and fatty acid composition of Temora longicornis females and different food species during the experiment

Temora longicornis, a dominant calanoid copepod species in the North Sea, is characterised by low lipid reserves and high biomass turnover rates. To survive and reproduce successfully, this species needs continuous food supply and thus requires a highly flexible digestive system to exploit various food sources. Information on the capacity of digestive enzymes is scarce and therefore the aim of our study was to investigate the enzymatic capability to respond to quickly changing nutritional conditions. We conducted two feeding experiments with female T. longicornis from the southern North Sea off Helgoland. In the first experiment in 2005, we tested how digestive enzyme activities and enzyme patterns as revealed by substrate SDS-PAGE (sodium dodecylsulfate polyacrylamide gel electrophoresis) responded to changes in food composition. Females were incubated for three days fed ad libitum with either the heterotrophic dinoflagellate Oxyrrhis marina or the diatom Thalassiosira weissflogii. At the beginning and at the end of the experiment, copepods were deep-frozen for analyses. The lipolytic enzyme activity did not change over the course of the experiment but the enzyme patterns did, indicating a distinct diet-induced response. In a second experiment in 2008, we therefore focused on the enzyme patterns, testing how fast changes occur and whether feeding on the same algal species leads to similar patterns. In this experiment, we kept the females for 4 days at surplus food while changing the algal food species daily. At day 1, copepods were offered O. marina. On day 2, females received the cryptophycean Rhodomonas baltica followed by T. weissflogii on day 3. On day 4 copepods were again fed with O. marina. Each day, copepods were frozen for analysis by means of substrate SDS-PAGE. This showed that within 24 h new digestive enzymes appeared on the electrophoresis gels while others disappeared with the introduction of a new food species, and that the patterns were similar on day 1 and 4, when females were fed with O. marina. In addition, we monitored the fatty acid compositions of the copepods, and this indicated that specific algal fatty acids were quickly incorporated. With such short time lags between substrate availability and enzyme response, T. longicornis can successfully exploit short-term food sources and is thus well adapted to changes in food availability, as they often occur in its natural environment due seasonal variations in phyto- and microzooplankton distribution.

Molecular and biochemical analyses of wounding in mesocarp of peach (Prunus persica L. Batsch) ripe fruits

The physiological and molecular responses of ripe fruit to wounding were evaluated in two peach (Prunus persica) varieties ('Glohaven', GH, melting and 'BigTop', BT, slow melting nectarine) by comparing mesocarp samples from wedges (as in minimal processing) and whole fruit as the control. Slight differences between the two varieties were detected in terms of ethylene production, whereas total phenol and flavonoid concentrations, and PPO and POD enzyme activities showed a general increase in wounded GH but not in BT. This was associated with the better appearance of the BT wedges at the end of the experimental period (72 h). Microarray (genome-wide ?PEACH3.0) analysis revealed that a total number of 2218 genes were differentially expressed (p < 0.01, log2 fold change expression ratio >1 or <-1) in GH 24 h after wounding compared to the control. This number was much lower (1208) in BT. According to the enrichment analysis, cell wall, plasma membrane, response to stress, secondary metabolic processes, oxygen binding were the GO categories over-represented among the GH up-regulated genes, whereas plasma membrane and response to endogenous stimulus were the categories over-represented among the down-regulated genes. Only 32 genes showed a common expression trend in the two varieties 24 h after wounding, whereas a total of 512 genes (with highly represented transcription factors), displayed opposite behavior. Quantitative RT-PCR analysis confirmed the microarray data for 18 out of a total of 20 genes selected. Specific WRKY, AP2/ERF and HSP20 genes were markedly up-regulated in wounded GH, indicating the activation of regulatory and signaling mechanisms probably related to different hormone categories. Compared to BT, the expression of specific genes involved in phenylpropanoid and triterpenoid biosynthetic pathways showed a more pronounced induction in GH, highlighting the difference between the two peach varieties in terms of molecular responses to wounding in the mesocarp tissue.

Temperature tolerance of different larval stages of the spider crab Hyas araneus exposed to elevated seawater PCO2

Exposure to elevated seawater PCO2 limits the thermal tolerance of crustaceans but the underlying mechanisms have not been comprehensively explored. Larval stages of crustaceans are even more sensitive to environmental hypercapnia and possess narrower thermal windows than adults. In a mechanistic approach, we analysed the impact of high seawater CO2 on parameters at different levels of biological organization, from the molecular to the whole animal level. At the whole animal level we measured oxygen consumption, heart rate and activity during acute warming in zoea and megalopa larvae of the spider crab Hyas araneus exposed to different levels of seawater PCO2. Furthermore, the expression of genes responsible for acid-base regulation and mitochondrial energy metabolism, and cellular responses to thermal stress (e.g. the heat shock response) was analysed before and after larvae were heat shocked byrapidly raising the seawater temperature from 10°C rearing temperature to 20°C. Zoea larvae showed a high heat tolerance, which decreased at elevated seawater PCO2, while the already low heat tolerance of megalopa larvae was not limited further by hypercapnic exposure. There was a combined effect of elevated seawater CO2 and heat shock in zoea larvae causing elevated transcript levels of heat shock proteins. In all three larval stages, hypercapnic exposure elicited an up-regulation of genes involved in oxidative phosphorylation, which was, however, not accompanied by increased energetic demands. The combined effect of seawater CO2 and heat shock on the gene expression of heat shock proteins reflects the downward shift in thermal limits seen on the whole animal level and indicates an associated capacity to elicit passive thermal tolerance. The up-regulation of genes involved in oxidative phosphorylation might compensate for enzyme activities being lowered through bicarbonate inhibition and maintain larval standard metabolic rates at high seawater CO2 levels. The present study underlines the necessity to align transcriptomic data with physiological responses when addressing mechanisms affected by an interaction of elevated seawater PCO2 and temperature extremes.

Response of three krill species to hypoxia and warming: An experimental approach to oxygen minimum zones expansion in coastal ecosystems

To understand the adaptation of euphausiid (krill) species to oxygen minimum zones (OMZ), respiratory response and stress experiments combining hypoxia/reoxygenation exposure with warming were conducted. Experimental krill species were obtained from the Antarctic (South Georgia area), the Humboldt Current system (HCS, Chilean coast), and the Northern California Current system (NCCS, Oregon). Euphausia mucronata from the HCS shows oxyconforming or oxygen partial pressure (pO2)-dependent respiration below 80% air saturation (18 kPa). Normoxic subsurface oxygenation in winter posed a "high oxygen stress" for this species. The NCCS krill, Euphausia pacifica, and the Antarctic krill, Euphausia superba maintain respiration rates constant down to low critical pO2 values of 6 kPa (30% air saturation) and 11 kPa (55% air saturation), respectively. Antarctic krill had the lowest antioxidant enzyme activities, but the highest concentrations of the molecular antioxidant glutathione (GSH) and was not affected by 6 h exposure to moderate hypoxia. Temperate krill species had higher SOD (superoxide dismutase) values in winter than in summer, which relate to higher winter metabolic rate (E. pacifica). In all species, antioxidant enzyme activities remained constant during hypoxic exposure at habitat temperature. Warming by 7°C above habitat temperature in summer increased SOD activities and GSH levels in E. mucronata (HCS), but no oxidative damage occurred. In winter, when the NCCS is well mixed and the OMZ is deeper, +4°C of warming combined with hypoxia represents a lethal condition for E. pacifica. In summer, when the OMZ expands upwards (100 m subsurface), antioxidant defences counteracted hypoxia and reoxygenation effects in E. pacifica, but only at mildly elevated temperature (+2°C). In this season, experimental warming by +4°C reduced antioxidant activities and the hypoxia combination again caused mortality of exposed specimens. We conclude that a climate change scenario combining warming and hypoxia represents a serious threat to E. pacifica and, as a consequence, NCCS food webs.

(Table 1) Polychlorinated biphenyl (PCB) and PCB metabolite concentrations in ringed seals (Phoca hispida) from Svalbard and the Baltic Sea

Polychlorinated biphenyls (PCBs) may induce activity of hepatic enzymes, mainly Phase I monooxygenases and conjugating Phase II enzymes, that catalyze the metabolism of PCBs leading to formation of metabolites and to potential adverse health effects. The present study investigates the concentration and pattern of PCBs, the induction of hepatic phase I and II enzymes, and the formation of hydroxy (OH) and methylsulfonyl (CH3SO2=MeSO2) PCB metabolites in two ringed seal (Phoca hispida) populations, which are contrasted by the degree of contamination exposure, that is, highly contaminated Baltic Sea (n = 31) and less contaminated Svalbard (n = 21). Phase I enzymes were measured as ethoxyresorufin-O-deethylation (EROD), benzyloxyresorufin-O-dealkylation (BROD), methoxyresorufin-O-demethylation (MROD), and pentoxyresorufin-O-dealkylation (PROD) activities, and phase II enzymes were measured as uridine diphosphophate glucuronosyl transferase (UDPGT) and glutathione-S-transferase (GST). Geographical comparison, multivariate, and correlation analysis indicated that sum-PCB had a positive impact on Phase I enzyme and GST activities leading to biotransformation of group III (vicinal ortho-meta-H atoms and <=1 ortho-chlorine (Cl)) and IV PCBs (vicinal meta-para-H atoms and <=2 ortho-Cl). The potential precursors for the main OH-PCBs detected in plasma in the Baltic seals were group III PCBs. MeSO2-PCBs detected in liver were mainly products of group IV PCB metabolism. Both CYP1A- and CYP2B-like enzymes are suggested to be involved in the PCB biotransformation in ringed seals.

Prosome length as well as carbon and nitrogen content of Calanus finmarchicus from the North Atlantic collected during Maria S. Merian cruise MSM26 in spring 2013

The prosome length of copepods from each station was measured on board with a dissecting microscope equipped with an ocular micrometer. Individuals were placed in pre-weighed tin caps and dried for 48 h at 60°C on board. Dry samples were transferred to the AWI and weighed again. Copepod dry mass was then calculated as the difference between the empty weight and the weight of the tin cap containing one individual. The content of carbon (C) and nitrogen (N) then was analysed with a CN-analyser (EuroEA Element Analyser, Hekatech) with acetanilide as standard.

Protein content of Calanus finmarchicus from the North Atlantic collected during Maria S. Merian cruise MSM26 in spring 2013

For the determination of water-soluble protein content of C. finmarchicus of the different stations the Qubit® Protein Assay Kit (Invitrogen) was used. Analysis was performed with extracts of 10 copepods. Working solution was prepared with Qubit® protein reagent and Qubit® protein buffer (1:200). 190 µL working solution was pipetted into each well of a micro plate and 10 µL of sample or Qubit® protein standard (0, 200 and 400 ng/µL) was added. Solutions were mixed and incubated for 15 min at room temperature. Measurements were conducted with a micro plate reader (TriStar LB 941, Berthold Technologies) at 485 nm excitation and 590 nm emission, using the software MikroWin2000 (Berthold Technologies).

Impact of CO2 enrichment on organic matter dynamics during nutrient induced coastal phytoplankton blooms

A mesocosm experiment was conducted to investigate the impact of rising fCO2 on the build-up and decline of organic matter during coastal phytoplankton blooms. Five mesocosms (~38 m³ each) were deployed in the Baltic Sea during spring (2009) and enriched with CO2 to yield a gradient of 355-862 µatm. Mesocosms were nutrient fertilized initially to induce phytoplankton bloom development. Changes in particulate and dissolved organic matter concentrations, including dissolved high-molecular weight (>1 kDa) combined carbohydrates, dissolved free and combined amino acids as well as transparent exopolymer particles (TEP), were monitored over 21 days together with bacterial abundance, and hydrolytic extracellular enzyme activities. Overall, organic matter followed well-known bloom dynamics in all CO2 treatments alike. At high fCO2, higher dPOC:dPON during bloom rise, and higher TEP concentrations during bloom peak, suggested preferential accumulation of carbon-rich components. TEP concentration at bloom peak was significantly related to subsequent sedimentation of particulate organic matter. Bacterial abundance increased during the bloom and was highest at high fCO2. We conclude that increasing fCO2 supports production and exudation of carbon-rich components, enhancing particle aggregation and settling, but also providing substrate and attachment sites for bacteria. More labile organic carbon and higher bacterial abundance can increase rates of oxygen consumption and may intensify the already high risk of oxygen depletion in coastal seas in the future.