Responses of the metabolism of the larvae of Pocillopora damicornis to ocean acidification and warming

Ocean acidification and warming are expected to threaten the persistence of tropical coral reef ecosystems. As coral reefs face multiple stressors, the distribution and abundance of corals will depend on the successful dispersal and settlement of coral larvae under changing environmental conditions. To explore this scenario, we used metabolic rate, at holobiont and molecular levels, as an index for assessing the physiological plasticity of Pocillopora damicornis larvae from this site to conditions of ocean acidity and warming. Larvae were incubated for 6 hours in seawater containing combinations of CO2 concentration (450 and 950 µatm) and temperature (28 and 30°C). Rates of larval oxygen consumption were higher at elevated temperatures. In contrast, high CO2 levels elicited depressed metabolic rates, especially for larvae released later in the spawning period. Rates of citrate synthase, a rate-limiting enzyme in aerobic metabolism, suggested a biochemical limit for increasing oxidative capacity in coral larvae in a warming, acidifying ocean. Biological responses were also compared between larvae released from adult colonies on the same day (cohorts). The metabolic physiology of Pocillopora damicornis larvae varied significantly by day of release. Additionally, we used environmental data collected on a reef in Moorea, French Polynesia to provide information about what adult corals and larvae may currently experience in the field. An autonomous pH sensor provided a continuous time series of pH on the natal fringing reef. In February/March, 2011, pH values averaged 8.075±0.023. Our results suggest that without adaptation or acclimatization, only a portion of naïve Pocillopora damicornis larvae may have suitable metabolic phenotypes for maintaining function and fitness in an end-of-the century ocean.

13C NMR study of the effects of leptin treatment on kinetics of hepatic intermediary metabolism

The recent discovery of leptin receptors in peripheral tissue raises questions about which of leptin’s biological actions arise from direct effects of the hormone on extraneural tissues and what intracellular mechanisms are responsible for leptin’s effects on carbohydrate and lipid metabolism. The present study is focused on the action of leptin on hepatic metabolism. Nondestructive 13C NMR methodology was used to follow the kinetics of intermediary metabolism by monitoring flux of 13C-labeled substrate through several multistep pathways. In perfused liver from either ob/ob or lean mice, we found that acute treatment with leptin in vitro modulates pathways controlling carbohydrate flux into 13C-labeled glycogen, thereby rapidly enhancing synthesis by an insulin-independent mechanism. Acute treatment of ob/ob liver also caused a rapid stimulation of long-chain fatty acid synthesis from 13C-labeled acetyl-CoA by the de novo synthesis route. Chronic leptin treatment in vivo induced homeostatic changes that resulted in a tripling of the rate of glycogen synthesis via the gluconeogenic pathway from [2-13C]pyruvate in ob/ob mouse liver perfused in the absence of the hormone. Consistent with the 13C NMR results, leptin treatment of the ob/ob mouse in vivo resulted in significantly increased hepatic glycogen synthase activity. Chronic treatment with leptin in vivo exerted the opposite effect of acute treatment in vitro and markedly decreased hepatic de novo synthesis of fatty acids in ob/ob mouse liver. In agreement with the 13C NMR findings, activities of hepatic acetyl-CoA carboxylase and fatty acid synthase were significantly reduced by chronic treatment of the ob/ob mouse with leptin. Our data represent a demonstration of direct effects of leptin in the regulation of metabolism in the intact functioning liver.

Protein C-Mannosylation Is Enzyme-catalysed and Uses Dolichyl-Phosphate-Mannose as a Precursor

C-mannosylation of Trp-7 in human ribonuclease 2 (RNase 2) is a novel kind of protein glycosylation that differs fundamentally from N- and O-glycosylation in the protein-sugar linkage. Previously, we established that the specificity determinant of the acceptor substrate (RNase 2) consists of the sequence W-x-x-W, where the first Trp becomes C-mannosylated. Here we investigated the reaction with respect to the mannosyl donor and the involvement of a glycosyltransferase. C-mannosylation of Trp-7 was reduced 10-fold in CHO (Chinese hamster ovary) Lec15 cells, which are deficient in dolichyl-phosphate-mannose (Dol-P-Man) synthase activity, compared with wild-type cells. This was not a result of a decrease in C-mannosyltransferase activity. Rat liver microsomes were used to C-mannosylate the N-terminal dodecapeptide from RNase 2 in vitro, with Dol-P-Man as the donor. This microsomal transferase activity was destroyed by heat and protease treatment, and displayed the same acceptor substrate specificity as the in vivo reaction studied previously. The C-C linkage between the indole and the mannosyl moiety was demonstrated by tandem electrospray mass spectrometry analysis of the product. GDP-Man, in the presence of Dol-P, functioned as a precursor in vitro with membranes from wild-type but not CHO Lec15 cells. In contrast, with Dol-P-Man both membrane preparations were equally active. It is concluded that a microsomal transferase catalyses C-mannosylation of Trp-7, and that the minimal biosynthetic pathway can be defined as: Man –> –> GDP-Man –> Dol-P-Man –> (C2-Man-)Trp.

Differential Regulation of Sugar-Sensitive Sucrose Synthases by Hypoxia and Anoxia Indicate Complementary Transcriptional and Posttranscriptional Responses1

The goal of this research was to resolve the hypoxic and anoxic responses of maize (Zea mays) sucrose (Suc) synthases known to differ in their sugar regulation. The two maize Suc synthase genes, Sus1 and Sh1, both respond to sugar and O2, and recent work suggests commonalities between these signaling systems. Maize seedlings (NK508 hybrid, W22 inbred, and an isogenic sh1-null mutant) were exposed to anoxic, hypoxic, and aerobic conditions (0, 3, and 21% O2, respectively), when primary roots had reached approximately 5 cm. One-centimeter tips were excised for analysis during the 48-h treatments. At the mRNA level, Sus1 was rapidly up-regulated by hypoxia (approximately 5-fold in 6 h), whereas anoxia had less effect. In contrast, Sh1 mRNA abundance increased strongly under anoxia (approximately 5-fold in 24 h) and was much less affected by hypoxia. At the enzyme level, total Suc synthase activity rose rapidly under hypoxia but showed little significant change during anoxia. The contributions of SUS1 and SH1 activities to these responses were dissected over time by comparing the sh1-null mutant with the isogenic wild type (Sus+, Sh1+). Sh1-dependent activity contributed most markedly to a rapid protein-level response consistently observed in the first 3 h, and, subsequently, to a long-term change mediated at the level of mRNA accumulation at 48 h. A complementary midterm rise in SUS1 activity varied in duration with genetic background. These data highlight the involvement of distinctly different genes and probable signal mechanisms under hypoxia and anoxia, and together with earlier work, show parallel induction of “feast and famine” Suc synthase genes by hypoxia and anoxia, respectively. In addition, complementary modes of transcriptional and posttranscriptional regulation are implicated by these data, and provide a mechanism for sequential contributions from the Sus1 and Sh1 genes during progressive onset of naturally occurring low-O2 events.

Epicuticular Wax Accumulation and Fatty Acid Elongation Activities Are Induced during Leaf Development of Leeks1

Epicuticular wax production was evaluated along the length of expanding leek (Allium porrum L.) leaves to gain insight into the regulation of wax production. Leaf segments from the bottom to the top were analyzed for (a) wax composition and load; (b) microsomal fatty acid elongase, plastidial fatty acid synthase, and acyl-acyl carrier protein (ACP) thioesterase activities; and (c) tissue and cellular morphological changes. The level of total wax, which was low at the bottom, increased 23-fold along the length of the leaf, whereas accumulation of the hentriacontan-16-one increased more than 1000-fold. The onset of wax accumulation was not linked to cell elongation but, rather, occurred several centimeters above the leaf base. Peak microsomal fatty acid elongation activity preceded the onset of wax accumulation, and the maximum fatty acid synthase activity was coincident with the onset. The C16:0- and C18:0-ACP-hydrolyzing activities changed relatively little along the leaf, whereas C18:1-ACP-hydrolyzing activity increased slightly prior to the peak elongase activity. Electron micrographic analyses revealed that wax crystal formation was asynchronous among cells in the initial stages of wax deposition, and morphological changes in the cuticle and cell wall preceded the appearance of wax crystals. These studies demonstrated that wax production and microsomal fatty acid elongation activities were induced within a defined and identifiable region of the expanding leek leaf and provide the foundation for future molecular studies.

Transport, Compartmentation, and Metabolism of Homoserine in Higher Plant Cells : Carbon-13- and Phosphorus-31-Nuclear Magnetic Resonance Studies

The transport, compartmentation, and metabolism of homoserine was characterized in two strains of meristematic higher plant cells, the dicotyledonous sycamore (Acer pseudoplatanus) and the monocotyledonous weed Echinochloa colonum. Homoserine is an intermediate in the synthesis of the aspartate-derived amino acids methionine, threonine (Thr), and isoleucine. Using 13C-nuclear magnetic resonance, we showed that homoserine actively entered the cells via a high-affinity proton-symport carrier (Km approximately 50–60 μm) at the maximum rate of 8 ± 0.5 μmol h−1 g−1 cell wet weight, and in competition with serine or Thr. We could visualize the compartmentation of homoserine, and observed that it accumulated at a concentration 4 to 5 times higher in the cytoplasm than in the large vacuolar compartment. 31P-nuclear magnetic resonance permitted us to analyze the phosphorylation of homoserine. When sycamore cells were incubated with 100 μm homoserine, phosphohomoserine steadily accumulated in the cytoplasmic compartment over 24 h at the constant rate of 0.7 μmol h−1 g−1 cell wet weight, indicating that homoserine kinase was not inhibited in vivo by its product, phosphohomoserine. The rate of metabolism of phosphohomoserine was much lower (0.06 μmol h−1 g−1 cell wet weight) and essentially sustained Thr accumulation. Similarly, homoserine was actively incorporated by E. colonum cells. However, in contrast to what was seen in sycamore cells, large accumulations of Thr were observed, whereas the intracellular concentration of homoserine remained low, and phosphohomoserine did not accumulate. These differences with sycamore cells were attributed to the presence of a higher Thr synthase activity in this strain of monocot cells.

Cells expressing the DG42 gene from early Xenopus embryos synthesize hyaluronan.

DG42 is one of the main mRNAs expressed during gastrulation in embryos of Xenopus laevis. Here we demonstrate that cells expressing this mRNA synthesize hyaluronan. The cloned DG42 cDNA was expressed in rabbit kidney (RK13) and human osteosarcoma (tk-) cells using a vaccinia virus system. Lysates prepared from infected cells were incubated in the presence of UDP-N-acetylglucosamine and UDP-[14C]glucuronic acid. This yielded a glycosaminoglycan with a molecular mass of about 200,000 Da. Formation of this product was only observed in the presence of both substrates. The glycosaminoglycan could be digested with testicular hyaluronidase and with Streptomyces hyaluronate lyase but not with Serratia chitinase. Hyaluronan synthase activity could also be detected in homogenates of early Xenopus embryos, and the activity was found to correlate with the expression of DG42 mRNA at different stages of development. Synthesis of hyaluronan is thus an early event after midblastula transition, indicating its importance for the ensuing cell movements in the developing embryo. Our results are at variance with a recent report (Semino, C. E. & Robbins, P. W. (1995) Proc. Natl. Acad. Sci. USA 92, 3498-3501) that DG42 codes for an enzyme that catalyzes the synthesis of chitin-like oligosaccharides.

Homologs of the Xenopus developmental gene DG42 are present in zebrafish and mouse and are involved in the synthesis of Nod-like chitin oligosaccharides during early embryogenesis.

The Xenopus developmental gene DG42 is expressed during early embryonic development, between the midblastula and neurulation stages. The deduced protein sequence of Xenopus DG42 shows similarity to Rhizobium Nod C, Streptococcus Has A, and fungal chitin synthases. Previously, we found that the DG42 protein made in an in vitro transcription/translation system catalyzed synthesis of an array of chitin oligosaccharides. Here we show that cell extracts from early Xenopus and zebrafish embryos also synthesize chitooligosaccharides. cDNA fragments homologous to DG42 from zebrafish and mouse were also cloned and sequenced. Expression of these homologs was similar to that described for Xenopus based on Northern and Western blot analysis. The Xenopus anti-DG42 antibody recognized a 63-kDa protein in extracts from zebrafish embryos that followed a similar developmental expression pattern to that previously described for Xenopus. The chitin oligosaccharide synthase activity found in extracts was inactivated by a specific DG42 antibody; synthesis of hyaluronic acid (HA) was not affected under the conditions tested. Other experiments demonstrate that expression of DG42 under plasmid control in mouse 3T3 cells gives rise to chitooligosaccharide synthase activity without an increase in HA synthase level. A possible relationship between our results and those of other investigators, which show stimulation of HA synthesis by DG42 in mammalian cell culture systems, is provided by structural analyses to be published elsewhere that suggest that chitin oligosaccharides are present at the reducing ends of HA chains. Since in at least one vertebrate system hyaluronic acid formation can be inhibited by a pure chitinase, it seems possible that chitin oligosaccharides serve as primers for hyaluronic acid synthesis.

cGMP mediates the vascular and platelet actions of nitric oxide: confirmation using an inhibitor of the soluble guanylyl cyclase.

The L-arginine:nitric oxide (NO) pathway is believed to exert many of its physiological effects via stimulation of the soluble guanylyl cyclase (SGC); however, the lack of a selective inhibitor of this enzyme has prevented conclusive demonstration of this mechanism of action. We have found that the compound 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) inhibits the elevation of cGMP induced by the NO donor S-nitroso-DL-penicillamine in human platelets and rat vascular smooth muscle (IC50 = 10-60 nM and <10 nM, respectively) and that this is accompanied by prevention of the platelet inhibitory and vasodilator actions of NO donors. ODQ also inhibited the antiaggregatory action of NO generated by the platelets but did not affect the action of prostacyclin or that of a cGMP mimetic. In addition, ODQ inhibited the vasodilator actions of endogenously released NO and of NO generated after induction of NO synthase in vascular preparations. It did not, however, affect the increase in vascular smooth muscle cGMP or the dilatation induced by atrial natriuretic factor. ODQ had no effect on NO synthase activity, nor did it react with NO. It did, however, potently (IC50 approximately 10 nM) inhibit the activity of the SGC in cytosol obtained from crude extract of rat aortic smooth muscle. Thus ODQ prevents the actions of NO on platelets and vascular smooth muscle through its potent inhibitory effect on the SGC.

Sphingolipid synthesis as a target for chemotherapy against malaria parasites.

The human malaria parasite Plasmodium falciparum contains sphingomyelin synthase in its Golgi apparatus and in a network of tubovesicular membranes in the cytoplasm of the infected erythrocyte. Palmitoyl and decanoyl analogues of 1-phenyl-2-acylamino-3-morpholino-1-propanol inhibit the enzyme activity in infected erythrocytes. An average of 35% of the activity is extremely sensitive to these drugs and undergoes a rapid, linear decrease at drug concentrations of 0.05-1 microM. The remaining 65% suffers a slower linear inhibition at drug concentrations ranging from 25 to 500 microM. Evidence is presented that inhibition of the sensitive fraction alone selectively disrupts the appearance of the interconnected tubular network in the host cell cytoplasm, without blocking secretory development at the parasite plasma membrane or in organelles within the parasite, such as the Golgi and the digestive food vacuole. This inhibition also blocks parasite proliferation in culture, indicating that the sensitive sphingomyelin synthase activity as well as the tubovesicular network may provide rational targets for drugs against malaria.

L-arginine attenuates cardiovascular impairment in DOCA-salt hypertensive rats

Nitric oxide (NO) is essential for normal function of the cardiovascular system. This study has determined whether chronic administration of L-arginine, the biological precursor of NO, attenuates the development of structural and functional changes in hearts and blood vessels of deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Uninephrectomized rats treated with DOCA (25 mg every 4th day sc) and 1% NaCl in the drinking water for 4 wk were treated with L-arginine (5% in food, 3.4 +/- 0.3 g.kg body wt(-1).day(-1)). Changes in cardiovascular structure and function were determined by echocardiography, microelectrode studies, histology, and studies in isolated hearts and thoracic aortic rings. DOCA-salt hypertensive rats developed hypertension, left ventricular hypertrophy with increased left ventricular wall thickness and decreased ventricular internal diameter, increased inflammatory cell infiltration, increased ventricular interstitial and perivascular collagen deposition, increased passive diastolic stiffness, prolonged action potential duration, increased oxidative stress, and inability to increase purine efflux in response to an increased workload. L-Arginine markedly attenuated or prevented these changes and also normalized the reduced efficacy of norepinephrine and acetylcholine in isolated thoracic aortic rings of DOCA-salt hypertensive rats. This study suggests that a functional NO deficit in blood vessels and heart due to decreased NO synthase activity or increased release of reactive oxygen species such as superoxide may be a key change initiating many aspects of the cardiovascular impairment observed in DOCA-salt hypertensive rats. These changes can be prevented or attenuated by administration of L-arginine.

Aspects of signal transduction in the gastrointestinal tract

This study was undertaken to increase knowledge of the mechanisms of inter- and intracellular signalling in the gastrointestinal tract. Specific aims were: to use cell lines to elucidate factors affecting growth of gastric cells, to investigate the distribution and aspects of function of isoforms of protein kinase C in a gastric cell line and in the rat gastrointestinal tract and to determine the presence and regulation of nitric oxide synthase in gastrointestinal tissues from the rat and in cell lines. The gastric cancer cell line HGT-1 was used to investigate control of growth. Increases in cell number were found to be dependent on the seeding density of the cells. In cells plated at low density insulin, epidermal growth factor and gastrin all increased cell number. Gastrin produced a bell-shaped dose response curve with a maximum activity at 5nM. No effect of gastrin was apparent in cells plated at high density. α and β isoforms of protein kinase C were found, by immunoblotting procedures, to be widespread in the gastrointestinal tract of the rat, but protein kinase Cε was confined to the gastric mucosa and gastrointestinal smooth muscle. HGT-1 cells contained protein kinase C α and ε but β or γ were not detected. Preincubation of HGT-1 cells for 24h with 1μM phorbol-12,13-dibutyrate down-regulated protein kinase C α but not ε. The inhibition by the activator of protein kinase C, 12-O-tetradecanoylphorbol 13-acetate (TPA) of the histamine-stimulated increase in cAMP in HGT-1 cells was down regulated by phorbol-12,13-dibutyrate. Inhibition of histamine-stimulation of adenylate cyclase by TPA was Ca2+-dependent and inhibited by the addition of an antibody to protein kinase C α. A role for protein kinase C α in modulating the effect of histamine on adenylate cyclase in HGT-1 cells is suggested. No nitric oxide synthase activity was detected in the gastrointestinal cell lines HGT-l, MKN-45 or CaCo-2. Ca2+-dependent nitric oxide synthase activity was observed in the gastric mucosa and the gastrointestinal smooth muscle from stomach to colon. The gastric: mucosal enzyme was soluble and showed half-maximal activity at 400nM Ca2+. Pretreatment of rats with endotoxin (3mg/kg body weight) induced nitric oxide synthase activity in both jejunal, ileal and colonic mucosa and muscle. A major portion of the induced activity in ileal and colonic mucosa was Ca2+-independent. Nitric oxide synthase activity in a high-density fraction of gastric mucosal cells was inhibited in a dose-dependent fashion by L-nitroarginine, NG-monomethyl-L-arginine, trifluoperazine and L-canavanine (in descending order of potency). Preincubation with okadaic acid and addition of ATPlMg2+ to the homogenisation buffer inhibited enzyme activity, which implies that phosphorylation inhibits gastric mucosal nitric oxide synthase.

Production and action of local mediators in the rat gastric mucosa

This study concerns the production and action of the local mediators nitric oxide (NO) and prostaglandin E2 (PGE2) in the rat gastric mucosa. The major objectives were: (i) to determine which mucosal cell type(s) contained NO synthase activity, (ii) to establish the functional role(s) of NO in the gastric mucosa and (iii) to investigate regulation of gastric PGE2 production. Gastric mucosal cells were isolated by pronase digestion coupled with intermittent calcium chelation and were separated by either density-gradient centrifugation or by counterflow elutriation. The distribution of Ca2+ -dependent NO synthase activity, measured via the conversion of [14C]-L-arginine to [14C]-L- citrulline, paralleled the distribution of mucous cells in elutriated fractions. Pre-treatment of rats with lipopolysaccharide caused the induction of Ca2+ -independent NO synthase in the elutriator fractions enriched with mucous cells. Incubation of isolated cells with the NO donor isosorbide dinitrate (ISDN) produced a concentration-dependent increase in the guanosine 3',-5'-cyclic monophosphate (cGMP) content which was accompanied by a concentration-dependent increase in release of immunoreactive mucin. Intragastric administration of ISDN of dibutyryl cGMP in vivo increased the thickness of the mucus layer overlying the gastric mucosa. The NO donor S-nitroso-N-acetylpenicillamine (SNAP) produced a concentration-dependent inhibition (IC50 247 μM) of histamine-stimulated aminopyrine accumulation, a measure of secretory activity, in cell suspensions containing > 80% parietal cells. SNAP increased the cGMP content of the suspension but did not decrease cellular viability, glucose oxidation or adenosine 3',5'-cyclic monophosphate content. The inhibitory effect of SNAP was observed in permeabilised cells stimulated with ATP and was stereospecifically blocked by preincubation with Rp-8-bromoguanosine 3'-5'-monophosphorothioate, which inhibits activation of cGMP-dependent protein kinase. Stimulation of PGE2 release by bradykinin in a low density cell fraction, enriched with parietal cells and devoid of vascular endothelial cells and macrophages, involved a bradykinin B1 receptor. In summary, NO synthase activity is probably present in gastric mucous epithelial cells. NO may promote mucus secretion by elevation of cGMP. NO donors inhibit acid secretion at a specific site and their action may involve cGMP. The bradykinin B1 receptor is involved with PGE2 production in the gastric mucosa.

Experiment: Elevated temperature and PCO2 affect enzyme activities in differentially oxidative tissues of Notothenia rossii

Mitochondrial plasticity plays a central role in setting the capacity for acclimation of aerobic metabolism in ectotherms in response to environmental changes. We still lack a clear picture if and to what extent the energy metabolism and mitochondrial enzymes of Antarctic fish can compensate for changing temperatures or PCO2 and whether capacities for compensation differ between tissues. We therefore measured activities of key mitochondrial enzymes (citrate synthase (CS), cytochrome c oxidase (COX)) from heart, red muscle, white muscle and liver in the Antarctic fish Notothenia rossii after warm- (7 °C) and hypercapnia- (0.2 kPa CO2) acclimation vs. control conditions (1 °C, 0.04 kPa CO2). In heart, enzymes showed elevated activities after cold-hypercapnia acclimation, and a warm-acclimation-induced upward shift in thermal optima. The strongest increase in enzyme activities in response to hypercapnia occurred in red muscle. In white muscle, enzyme activities were temperature-compensated. CS activity in liver decreased after warm-normocapnia acclimation (temperature-compensation), while COX activities were lower after cold- and warm-hypercapnia exposure, but increased after warm-normocapnia acclimation. In conclusion, warm-acclimated N. rossii display low thermal compensation in response to rising energy demand in highly aerobic tissues, such as heart and red muscle. Chronic environmental hypercapnia elicits increased enzyme activities in these tissues, possibly to compensate for an elevated energy demand for acid-base regulation or a compromised mitochondrial metabolism, that is predicted to occur in response to hypercapnia exposure. This might be supported by enhanced metabolisation of liver energy stores. These patterns reflect a limited capacity of N. rossii to reorganise energy metabolism in response to rising temperature and PCO2.