Altered cholesterol metabolism in a hypercholesterolemia -resistant rabbit colony

A colony of rabbits has been developed at the University of Texas Medical School at Houston that is resistant to dietary-induced hypercholesterolemia. The liver of resistant rabbits had higher levels of ($\sp{125}$I) $\beta$-VLDL binding and 3-hydroxy-3-methylglutaryl (HMGCoA) reductase activity, but lower acyl coenzyme A:cholesterol acyltransferase (ACAT) activity than normal rabbits. Direct quantitation of intracellular cholesterol content of the liver revealed that the resistant rabbits had $<$10% of the intracellular free cholesterol present in normal rabbits. Fibroblasts isolated from normal and resistant rabbits exhibited differences in ($\sp{125}$I) LDL binding, HMGCoA reductase activity and ACAT activity that were similar to those found in the liver. No structural differences were found in the LDL receptor of normal and resistant fibroblasts that would account for the increased binding capacity of the resistant cells. The regulation of LDL receptor levels by exogenous oxygenated sterols was similar in normal and resistant fibroblasts. The regulation of LDL receptor binding capacity by LDL was attenuated in the resistant compared to normal fibroblasts, suggesting that the resistant fibroblasts have an alternate pathway for processing lipoprotein-derived cholesterol. Sterol-balance studies revealed that the cholesterol-fed resistant rabbits increased lithocholic acid excretion compared to the basal state, and had higher levels of deoxycholic acid excretion than cholesterol-fed normal rabbits. In addition, the specific activity and mRNA levels of cholesterol 7$\alpha$-hydroxylase (C7$\alpha$H) were higher in resistant rabbits than normal rabbits, suggesting that the increased bile acid excretion was due to an increase in bile acid synthesis. Increased clearance of cholesterol relieves the negative feedback inhibition cholesterol exerts on expression of the LDL receptor. The number of cell surface LDL receptors is then increased in resistant rabbits and allows rapid clearance of lipoproteins from the plasma compartment, thereby reducing plasma cholesterol levels. The low intracellular cholesterol level also relieves the negative feedback inhibition cholesterol exerts on HMGCoA reductase. Increased synthesis of cholesterol from acetate provides cells with cholesterol for bile acid synthesis and/or homeostasis. The activity of ACAT is then decreased due to the flux of cholesterol through the bile acid synthetic pathways. ^

Characterization of the cholesterol 7$\alpha$-hydroxylase promoter in hypercholesterolemia -resistant rabbits

Our laboratory has developed and partially characterized a strain of New Zealand white rabbits that are resistant to the hypercholesterolemia which typically occurs in normal rabbits when fed a cholesterol-enriched diet. This phenotype is most likely attributed to an increase in bile acid excretion by hypercholesterolemia-resistant (CRT) rabbits as a result of elevated enzyme activity of cholesterol 7$\alpha$-hydroxylase (C7$\alpha$H), the rate-limiting enzyme in bile acid synthesis. Northern analysis revealed that CRT rabbits, in comparison to normal rabbits, have a 7-fold greater steady-state C7$\alpha$H mRNA levels irrespective of dietary regimen. The C7$\alpha$H gene in both phenotypes was determined to be a single copy gene. The hypothesis was that the elevated C7$\alpha$H mRNA levels in CRT rabbits, in comparison to normal animals, was due to an increase in the transcription rate of the C7$\alpha$H gene as a result of a mutation in a cis-acting element and/or a trans-acting factor within the hepatocyte. To isolate the C7$\alpha$H gene from both normal and CRT rabbits, genomic libraries were prepared from both phenotypes into $\lambda$GEM12 vectors using conventional techniques. Three CRT and one normal phage clones that contained the C7$\alpha$H gene were identified by screening the library with a series of probes located within different exons of the C7$\alpha$H cDNA. Sequencing analysis confirmed that approximately 1100 bp of the C7$\alpha$H 5'-flanking region from both normal and CRT phenotypes was identical. The increase in C7$\alpha$H mRNA levels was not attributed to a cis-acting mutation within this region. Liver nuclear extracts were prepared from normal and CRT rabbits maintained either on a basal or 0.25% cholesterol-enriched diet and incubated with several radiolabeled DNA fragments from the C7$\alpha$H gene. A 37 basepair region, located between nucleotides $-$452 to $-$416 was identified that had altered binding patterns between normal and CRT rabbits as a function of diet. Two additional regions, $-$747 to $-$575 and $-$580 to $-$442, produced banding patterns which were identical, irrespective of phenotype or diet. In conclusion, these studies suggested that the increase in C7$\alpha$H mRNA in CRT rabbits was due to differences in binding of a cholesterol-responsive transcription factor to the C7$\alpha$H promoter. ^

Metabolism of mesozooplankton in the North-Eastern Aegean Sea during SES_GR2 in October 2008

The dataset is based on samples taken during October 2008 in the North-Eastern Aegean Sea. NH4 excretion rate: Mesozooplankton is collected by vertical tows within the Black sea water body mass layer in the NE Aegean, using a WP-2 200 µm net equipped with a large non-filtering cod-end (10 l). Macrozooplankton organisms are removed using a 2000 µm net. A few unsorted animals (approximately 100) are placed inside 8 bottles of 350 or 650 ml filled with GF/F or 0.2 µm Nucleopore filtered seawater and then on a wheell at dim light and maintaining the in situ temperature. 4 bottles without animals are used as control. After 24hours bottles are opened and water samples taken for NH4 chemical analysis. Then the bottle content is filtered on pre-combusted preweighted CF/F filters, which are then dried at 60 C and weighted. Calculations are made as described by Ikeda et al. (2000). Samples for the NH4 determination were collected in pre-cleaned 50 ml Duran bottles and analysed onboard immediately after collection. Ammonium concentration was measured on a Perkin Elmer Lambda 25 UV/VIS Spectrometer according to the method of Koroleff (1970). PO4 excretion rate: Mesozooplankton is collected by vertical tows within the Black sea water body mass layer in the NE Aegean, using a WP-2 200 µm net equipped with a large non-filtering cod-end (10 l). Macrozooplankton organisms are removed using a 2000 µm net. A few unsorted animals (approximately 100) are placed inside 8 bottles of 350 or 650 ml filled with GF/F or 0.2 µm Nucleopore filtered seawater and then on a wheell at dim light and maintaining the in situ temperature. 4 bottles without animals are used as control. After 24hours bottles are opened and water samples taken for PO4 chemical analysis. Then the bottle content is filtered on pre-combusted preweighted CF/F filters, which are then dried at 60 C and weighted. Calculations are made as described by Ikeda et al. (2000). Samples for the determination of PO4 were collected in pre-cleaned 50 ml polyethylene volumetric tubes and analysed on board immediately after collection. PO4 concentration was measured on a Perkin Elmer Lambda 25 UV/VIS Spectrometer following the protocol of Murphy and Riley (1962). O2 consumption rate: Mesozooplankton is collected by vertical tows within the Black sea water body mass layer in the NE Aegean, using a WP-2 200 µm net equipped with a large non-filtering cod-end (10 l). Macrozooplankton organisms are removed using a 2000 µm net. A few unsorted animals (approximately 100) are placed inside 8 bottles of 350 or 650 ml filled with GF/F or 0.2 µm Nucleopore filtered seawater and then on a wheell at dim light and maintaining the in situ temperature. 4 bottles without animals are used as control. After 24hours bottles are opened and water samples taken for O2 chemical analysis. Then the bottle content is filtered on pre-combusted preweighted CF/F filters, which are then dried at 60 C and weighted. Calculations are made as described by Ikeda et al. (2000). For the dissolved O2 determination, the samples were fixed immediately after collection and analysed with the Winkler method as modified by Carpenter (1965a and 1965b). Carbon specific CO2 respiration rate: O2 consumption rate was converted to CO2 production using a RQ value of 0.87 (Mayzaud et al. 2005). Conversion of mesozooplankton dry weight to carbon was done using the % of carbon content measured in the same station from the SESAME dataset of zooplankton biomass. Carbon specific NH4 excretion rate: Conversion of mesozooplankton dry weight to carbon was done using the % of carbon content measured in the same station from the SESAME dataset of zooplankton biomass. Carbon specific PO4 excretion rate: Conversion of mesozooplankton dry weight to carbon was done using the % of carbon content measured in the same station from the SESAME dataset of zooplankton biomass.

Seawater carbonate chemistry, calcification and respiration duirng experiments with a pteropod Creseis acicula, 2010

Among marine calcifiers, shelled pteropods are expected to be particularly sensitive to ocean acidification, generated by the uptake of anthropogenic CO2 by the ocean, and the associated decrease of the seawater saturation state with respect to aragonite (omega aragonite). The few available studies have mostly focused on polar species although pteropods are also important components of temperate and tropical ecosystems. It is also unknown which parameter of the carbonate system controls calcification. Specimens of the temperate Mediterranean species Creseis acicula were maintained under seven different conditions of the carbonate chemistry, obtained by manipulating pH and total alkalinity, with the goal to disentangle the effects of pH and omega aragonite. Respiration, excretion as well as rates of net and gross calcification were not directly affected by a decrease in pH but decreased significantly with a decrease of omega aragonite. The decrease of gross calcification rates is consistent with that reported for polar species. Although the organisms were apparently able to maintain gross calcification rates under slightly undersaturated aragonite conditions, the clear net dissolution signal observed below saturation suggests that they are not able to build a shell in seawater corrosive to aragonite. The decrease in respiration and excretion, and the low O:N molar ratio, could be due to the short time that the organisms were allowed to acclimatize to their new environment.

Haemolymph inorganic ion composition, physiology and behaviour dependent on temperature and ambient magnesium concentration in early life history stages of Paralomis granulosa (Decapoda, Anomura, Lithodidae)

Marine brachyuran and anomuran crustaceans are completely absent from the extremely cold (-1.8 °C) Antarctic continental shelf, but caridean shrimps are abundant. This has at least partly been attributed to low capacities for magnesium excretion in brachyuran and anomuran lithodid crabs ([Mg2+]HL = 20-50 mmol/L) compared to caridean shrimp species ([Mg2+]HL = 5-12 mmol/L). Magnesium has an anaesthetizing effect and reduces cold tolerance and activity of adult brachyuran crabs. We investigated whether the capacity for magnesium regulation is a factor that influences temperature-dependent activity of early ontogenetic stages of the Sub-Antarctic lithodid crab Paralomis granulosa. Ion composition (Na+, Mg2+, Ca2+, Cl-, [SO4]2-) was measured in haemolymph withdrawn from larval stages, the first and second juvenile instars (crabs I and II) and adult males and females. Magnesium excretion improved during ontogeny, but haemolymph sulphate concentration was lowest in the zoeal stages. Neither haemolymph magnesium concentrations nor Ca2+:Mg2+ ratios paralleled activity levels of the life stages. Long-term (3 week) cold exposure of crab I to 1 °C caused a significant rise of haemolymph sulphate concentration and a decrease in magnesium and calcium concentrations compared to control temperature (9 °C). Spontaneous swimming activity of the zoeal stages was determined at 1, 4 and 9 °C in natural sea water (NSW, [Mg2+] = 51 mmol/L) and in sea water enriched with magnesium (NSW + Mg2+, [Mg2+] = 97 mmol/L). It declined significantly with temperature but only insignificantly with increased magnesium concentration. Spontaneous velocities were low, reflecting the demersal life style of the zoeae. Heart rate, scaphognathite beat rate and forced swimming activity (maxilliped beat rate, zoea I) or antennule beat rate (crab I) were investigated in response to acute temperature change (9, 6, 3, 1, -1 °C) in NSW or NSW + Mg2+. High magnesium concentration reduced heart rates in both stages. The temperature-frequency curve of the maxilliped beat (maximum: 9.6 beats/s at 6.6 °C in NSW) of zoea I was depressed and shifted towards warmer temperatures by 2 °C in NSW + Mg2+, but antennule beat rate of crab I was not affected. Magnesium may therefore influence cold tolerance of highly active larvae, but it remains questionable whether the slow-moving lithodid crabs with demersal larvae would benefit from an enhanced magnesium excretion in nature.

ATPase activity, proton gradients and proton secretion inhibition during experiments with Sepia officinalis and Sepioteuthis lessoniana

The constraints of an active life in a pelagic habitat led to numerous convergent morphological and physiological adaptations that enable cephalopod molluscs and teleost fishes to compete for similar resources. Here, we show for the first time that such convergent developments are also found in the ontogenetic progression of ion regulatory tissues; as in teleost fish, epidermal ionocytes scattered on skin and yolk sac of cephalopod embryos appear to be responsible for ionic and acid-base regulation before gill epithelia become functional. Ion and acid-base regulation is crucial in cephalopod embryos, as they are surrounded by a hypercapnic egg fluid with a Pco2 between 0.2 and 0.4 kPa. Epidermal ionocytes were characterized via immunohistochemistry, in situ hybridization, and vital dye-staining techniques. We found one group of cells that is recognized by concavalin A and MitoTracker, which also expresses Na+/H+ exchangers (NHE3) and Na+-K+-ATPase. Similar to findings obtained in teleosts, these NHE3-rich cells take up sodium in exchange for protons, illustrating the energetic superiority of NHE-based proton excretion in marine systems. In vivo electrophysiological techniques demonstrated that acid equivalents are secreted by the yolk and skin integument. Intriguingly, epidermal ionocytes of cephalopod embryos are ciliated as demonstrated by scanning electron microscopy, suggesting a dual function of epithelial cells in water convection and ion regulation. These findings add significant knowledge to our mechanistic understanding of hypercapnia tolerance in marine organisms, as it demonstrates that marine taxa, which were identified as powerful acid-base regulators during hypercapnic challenges, already exhibit strong acid-base regulatory abilities during embryogenesis.

Primary productivity and phytoplankton pigment measurements in the North Sea

During the culmination of the phytoplankton spring bloom in the Fladen Ground area in April-Mai 1976, gross primary production was between 1500 and 2000 mg particulate C m**-2 day**-1, at a crop density (mainly diatoms of the genus Chaetoceros) of about 1500-3500 mg C m**-2. Estimates of the C:chlorophyll a ratio in living cells were much lower than those reported in the literature, possibly because part of what is measured as "chlorophyll a" by the common fluorometric method is associated with particles that are not reported as cells. Most of the dark 14C fixation during the bloom's climax was due to abiotic processes. Excretion of 14C-labeled carbohydrates did not account for a significant fraction of the total photosynthetic rate. The low crop after the bloom period, in June, corresponded with nutrient depletion of the euphotic zone. The low photosynthetic efficiency in June may have been a gross underestimate. The presence of relatively high concentrations of chlorophyll derivatives signifies that the algal crop was consumed by heterotrophs, but at a lower rate in April/May than during the June cruise when particularly high molar ratios of phaeophorbide a and phaeophytin a relative to chlorophyll a were found. The high respiratory rate relative to autotrophic production in June manifested itself also in high dark 14C fixation values. The high concentration of phaeophorbide a in the upper 40 m and its scarcity below this depth during the spring bloom climax in April/May implies that copepod grazing at that time took place principally in the euphotic zone. The remarkably high concentration of chlorophyllide a in the surface layer during the bloom period indicates that the part of the crop that was destroyed by the grazers while eating was occasionally as high as the part that was actually ingested.

Abundance, biomass, ingestion rates and daily ratios of Salpa thompsoni individuals sampled during POLARSTERN cruise ANT-XVIII/5b to the Eastern Belingshausen Sea

Distribution, density, and feeding dynamics of the pelagic tunicate Salpa thompsoni have been investigated during the expedition ANTARKTIS XVIII/5b to the Eastern Bellingshausen Sea on board RV Polarstern in April 2001. This expedition was the German contribution to the field campaign of the Southern Ocean Global Ocean Ecosystems Dynamics Study (SO-GLOBEC). Salps were found at 31% of all RMT-8 and Bongo stations. Their densities in the RMT-8 samples were low and did not exceed 4.8 ind/m**2 and 7.4 mg C/m**2. However, maximum salp densities sampled with the Bongo net reached 56 ind/m**2 and 341 mg C/m**2. A bimodal salp length frequency distribution was recorded over the shelf, and suggested two recent budding events. This was also confirmed by the developmental stage composition of solitary forms. Ingestion rates of aggregate forms increased from 2.8 to 13.9 µg (pig)/ind/day or from 0.25 to 2.38 mg C/ind/day in salps from 10 to 40 mm oral-atrial length, accounting for 25-75% of body carbon per day. Faecal pellet production rates were on average 0.08 pellet/ind/h with a pronounced diel pattern. Daily individual egestion rates in 13 and 30 mm aggregates ranged from 0.6 to 4.8 µg (pig)/day or from 164 to 239 µg C/day. Assimilation efficiency ranged from 73 to 90% and from 65 to 76% in 13 and 30 mm aggregates, respectively. S. thompsoni exhibited similar ingestion and egestion rates previously estimated for low Antarctic (~50°S) habitats. It has been suggested that the salp population was able to develop in the Eastern Bellingshausen Sea due to an intrusion into the area of the warm Upper Circumpolar Deep Water

Seawater carbonate chemistry and biological processes of Mytilus edulis during experiments, 2010

Marine organisms are exposed to increasingly acidic oceans, as a result of equilibration of surface ocean water with rising atmospheric CO2 concentrations. In this study, we examined the physiological response of Mytilus edulis from the Baltic Sea, grown for 2 months at 4 seawater pCO2 levels (39, 113, 243 and 405 Pa/385, 1,120, 2,400 and 4,000 µatm). Shell and somatic growth, calcification, oxygen consumption and excretion rates were measured in order to test the hypothesis whether exposure to elevated seawater pCO2 is causally related to metabolic depression. During the experimental period, mussel shell mass and shell-free dry mass (SFDM) increased at least by a factor of two and three, respectively. However, shell length and shell mass growth decreased linearly with increasing pCO2 by 6-20 and 10-34%, while SFDM growth was not significantly affected by hypercapnia. We observed a parabolic change in routine metabolic rates with increasing pCO2 and the highest rates (+60%) at 243 Pa. excretion rose linearly with increasing pCO2. Decreased O:N ratios at the highest seawater pCO2 indicate enhanced protein metabolism which may contribute to intracellular pH regulation. We suggest that reduced shell growth under severe acidification is not caused by (global) metabolic depression but is potentially due to synergistic effects of increased cellular energy demand and nitrogen loss.

Trace element profiles of the sea anemone Anemonia viridis living nearby a natural CO2 vent

Ocean acidification (OA) is not an isolated threat, but acts in concert with other impacts on ecosystems and species. Coastal marine invertebrates will have to face the synergistic interactions of OA with other global and local stressors. One local factor, common in coastal environments, is trace element contamination. CO2 vent sites are extensively studied in the context of OA and are often considered analogous to the oceans in the next few decades. The CO2 vent found at Levante Bay (Vulcano, NE Sicily, Italy) also releases high concentrations of trace elements to its surrounding seawater, and is therefore a unique site to examine the effects of long-term exposure of nearby organisms to high pCO2 and trace element enrichment in situ. The sea anemone Anemonia viridis is prevalent next to the Vulcano vent and does not show signs of trace element poisoning/stress. The aim of our study was to compare A. viridis trace element profiles and compartmentalization between high pCO2 and control environments. Rather than examining whole anemone tissue, we analyzed two different body compartments-the pedal disc and the tentacles, and also examined the distribution of trace elements in the tentacles between the animal and the symbiotic algae. We found dramatic changes in trace element tissue concentrations between the high pCO2/high trace element and control sites, with strong accumulation of iron, lead, copper and cobalt, but decreased concentrations of cadmium, zinc and arsenic proximate to the vent. The pedal disc contained substantially more trace elements than the anemone's tentacles, suggesting the pedal disc may serve as a detoxification/storage site for excess trace elements. Within the tentacles, the various trace elements displayed different partitioning patterns between animal tissue and algal symbionts. At both sites iron was found primarily in the algae, whereas cadmium, zinc and arsenic were primarily found in the animal tissue. Our data suggests that A. viridis regulates its internal trace element concentrations by compartmentalization and excretion and that these features contribute to its resilience and potential success at the trace element-rich high pCO2 vent.