Net Synthesis Research Articles

Glycogen synthase kinase 3 inhibition improves insulin-stimulated glucose metabolism but not hypertension in high-fat-fed C57BL/6J mice

In the current study, the effect of a highly specific peptide inhibitor of glycogen synthase kinase 3 (GSK3) (L803-mts) on glucose metabolism and BP was examined in a high-fat (HF) fed mouse model of diabetes. C57/BL6J mice were placed on an HF diet for 3 months and treated with L803-mts for 20 days, following which glucose metabolism was examined by euglycaemic-hyperinsulinaemic clamp studies. BP and heart rate were measured by radio-telemetry. The HF mice were obese, with impaired glucose tolerance and high plasma insulin and leptin levels. L803-mts treatment significantly reduced the insulin levels and doubled the glucose infusion rate required to maintain a euglycaemic condition in the HF+L803-mts group compared with the HF group. Insulin failed to suppress the endogenous glucose production rate in the HF group while decreasing it by 75% in the HF+L803-mts group, accompanied by increased liver glycogen synthase activity and net hepatic glycogen synthesis. GSK3 inhibition also reduced peripheral insulin resistance. Plasma glucose disappearance rate increased by 60% in the HF+L803-mts group compared with the HF group. In addition, glucose uptake in heart and gastrocnemius muscle was markedly improved. Although mean arterial pressure increased following the HF diet, it did not change significantly during the 12 days of L803-mts treatment. These studies demonstrate that GSK3 inhibition improved hepatic and peripheral insulin resistance in a mouse model of HF-induced diabetes, but it failed to have an effect on BP. GSK3 may represent an important therapeutic target for insulin resistance.

Phosphatidylinositol 3-Kinase-dependent Modulation of Carnitine Palmitoyltransferase 1A Expression Regulates Lipid Metabolism during Hematopoietic Cell Growth

An abundant supply of extracellular nutrients is believed to be sufficient to suppress catabolism of cellular macromolecules. Here we show that, despite abundant extracellular nutrients, interleukin-3-deprived hematopoietic cells begin to catabolize intracellular lipids. Constitutive Akt activation blunts the increased beta-oxidation that accompanies growth factor withdrawal, and in growth factor-replete cells, phosphatidylinositol 3-kinase (PI3K) signaling is required to suppress lipid catabolism. Surprisingly, PI3K and Akt exert these effects by suppressing expression of the beta-oxidation enzyme carnitine palmitoyltransferase 1A (CPT1A). Cells expressing a short hairpin RNA against CPT1A fail to induce beta-oxidation in response to growth factor withdrawal and are unable to survive glucose deprivation. When CPT1A is constitutively expressed, growth factor stimulation fails to repress beta-oxidation. As a result, both net lipid synthesis and cell proliferation are diminished. Together, these results demonstrate that modulation of CPT1A expression by PI3K-dependent signaling is the major mechanism by which cells suppress beta-oxidation during anabolic growth.

The lipid-lowering effect of ezetimibe in pure vegetarians

Results of previous studies have shown that ezetimibe (10 mg/day) reduces LDL cholesterol in patients with mild hypercholesterolemia on a normal-cholesterol diet (dietary intake of 200-500 mg/day) by 16-22%. However, the LDL cholesterol-lowering effect of ezetimibe in subjects with an extremely low dietary cholesterol intake (vegetarians) has not been studied. We conducted a randomized, double-blind, placebo-controlled, two-phase crossover study in 18 healthy pure vegetarians to assess the effect of ezetimibe (10 mg/day) on plasma lipids, cholesterol absorption, and its synthesis. Treatment periods lasted 2 weeks each, with an intervening 2 week washout period. Fractional cholesterol absorption was determined using the continuous dual stable isotope feeding method. Mean dietary cholesterol intake in the pure vegetarians was extremely low and averaged 29.4 +/- 16.8 and 31.4 +/- 14.4 mg/day during the placebo and ezetimibe administration phases, respectively. Fractional cholesterol absorption during the placebo phase was 48.2 +/- 8.2% and was decreased by 58% during ezetimibe treatment to 20.2 +/- 6.2% (P < 0.001). This change in intestinal cholesterol absorption was followed by a significant reduction in LDL cholesterol of 17.3%. In individuals with extremely low dietary cholesterol intake, treatment with ezetimibe (10 mg/day) leads to a significant reduction of cholesterol absorption and a clinically relevant decrease of plasma LDL cholesterol, comparable to that of subjects with a normal dietary cholesterol intake. Thus, the lipid-lowering effect of ezetimibe is mediated mainly through a reduction of the absorption of endogenous (biliary) cholesterol.

Development of a recombinant strain of Bacillus thuringiensis subsp. kurstaki HD-73 that produces the endochitinase ChiA74

Bacillus thuringiensis subsp. kurstaki HD-73 was transformed with the homologous endochitinase gene chiA74 of B. thuringiensis subsp. kenyae LBIT-82 under the regulation of its own promoter and Shine-Dalgarno sequence. The plasmid, pEHchiA74, which harbors chiA74, was detected by southern blot analysis and showed high segregational stability when the recombinant strain was grown in a medium without antibiotic. The recombinant bacterium transformed with pEHchiA74 showed an improvement in chitinolytic activity three times that of the wild-type strain. Expression of ChiA74 did not have any deleterious effect on the crystal morphology and size, but sporulation and Cry1Ac production in rich medium (nutrient broth with glucose) was reduced by approximately 30%. No significant increase in the toxicity of the transformant bacterium toward Plutella xylostella was detected using the same amount of total protein. However, it is possible that ChiA74 synthesis compensated for the decrease in net Cry1Ac synthesis and toxicity observed with the recombinant strain.

Mesoscale variability in the metabolic balance of the Sargasso Sea

Net community production (NCP) experiments based on in vitro changes in dissolved oxygen were carried out in three mesoscale eddies investigated in the Sargasso Sea in the summer of 2004. NCP estimates ranged from negative to positive values, and the age and type of the sampled eddy were important factors responsible for this variability. Positive rates occurred in younger cyclones and in areas of eddy‐eddy interactions, whereas negative NCP rates were observed in anticyclones and older cyclone features that were decaying. This information was combined with a nine‐year data set from the BATS program and satellite altimeter data to assess the role of mesoscale eddies in the metabolic balance of the Sargasso Sea. Indirect NCP estimates (NCPe) were obtained by calculating the difference between 14C phytoplankton production and bacterial carbon demand rates. The results showed highly variable NCPe rates that spanned the range of direct NCP observations.

Phospholipid fatty acid pattern and d-glucose metabolism in muscles from ω3 fatty acid-depleted rats

A depletion in long-chain polyunsaturated ω3 fatty acids may affect fuel homeostasis. In such a perspective, the present study deals mainly with the in vitro fate of d-[U- 14C]glucose in hemidiaphragms, stretched soleus and plantaris muscle pieces obtained from normal and ω3-depleted rats (second generation) and incubated in the absence or presence of insulin. When so required, the ω3-depleted rats were injected 120 min before sacrifice with either a ω3 fatty acid-rich medium-chain triglyceride:fish oil emulsion (FO) or a control medium-chain triglyceride:olive oil emulsion (OO). The content of the soleus muscle in long-chain polyunsaturated ω3 fatty acids was severely decreased in the ω3-depleted rats, and modestly albeit significantly increased after injection of FO to these animals. In stretched soleus muscles from OO-injected ω3-depleted rats, the absolute values for glycogen synthesis measured in the absence or presence of insulin were about twice higher than in normal animals. In the OO-injected ω3-depleted rats, insulin augmented the output of 14C-labelled amino acids, whilst such was not the case in normal animals. These and other findings suggest a lower catabolism of d-glucose relative to the anabolic process of glycogen synthesis and a lower availability of endogenous amino acids in the muscles of ω3-depleted rats, as compared to those of control animals. The prior injection of FO to the ω3-depleted rats restored a normal value for the paired ratio between the output of 14C-labelled amino acids and acidic metabolites, but further increased glycogen net synthesis. It is proposed, therefore, that the perturbation of d-glucose metabolism in muscles from ω3-depleted rats involves a multifactorial determinism, only some of the concerned factors being susceptible to rapid correction after enrichment of cell phospholipids in long-chain polyunsaturated ω3 fatty acids.

Muscarinic M2Receptors Directly Activate Gq/11and GsG-Proteins

Muscarinic M(2) receptors preferentially couple with the G(i/o) class of G-proteins to inhibit cAMP synthesis. However, they can also stimulate net synthesis of cAMP and inositol phosphate (IP) accumulation. We investigated in intact Chinese hamster ovary (CHO) cells expressing human M(2) receptors (CHO-M(2) cells) whether direct interaction of M(2) receptors with G(s) and G(q/11) G-proteins is responsible for the latter effects. Suppression of the G(s)alpha subunit using RNA interference abolished stimulation of cAMP synthesis induced by 1 mM carbachol in both control and pertussis toxin-treated CHO-M(2) cells but had no effect on the inhibition of forskolin-stimulated cAMP synthesis. Carbachol stimulated accumulation of IP with an EC(50) of 79 microM. Removal of the G(q),G(11), or both alpha subunits reduced this response by 78, 54, and 92%, respectively, whereas suppression of the G(s)alpha subunit had no effect. Similar results obtained in CHO cells expressing M(1) receptors that preferentially couple with G(s) and G(q/11) G-proteins confirmed the efficiency of siRNA treatments. Stimulation of M(2) receptors in control and pertussis toxin-treated cells by a series of full agonists with respect to inhibition of adenylyl cyclase displayed different efficacies in stimulating IP accumulation. Carbachol, acetylcholine, and oxotremorine-M [N,N,N-trimethyl-4-(2-oxo-1-pyrolidinyl)-2-butyn-1-ammonium] behaved as full agonists, furmethide (N,N,N-trimethyl-2-furanmethammonium) and methylfurmethide [(5-methyl-2-furyl)methyltrimethylammonium] were partial agonists, and oxotremorine (1-[4-(1-pyrrolidinyl)-2-butynyl]-2-pyrrolidinone) had no effect. Our results provide direct evidence of M(2) receptor coupling with the alpha subunits of G(s) and G(q/11) G-proteins and demonstrate induction of multiple receptor conformational states dependent on both the concentration and the nature of the agonist used.

Treatment with HMGB1 inhibitors diminishes CTL-induced liver disease in HBV transgenic mice

Using hepatitis B virus (HBV) transgenic mice as recipients of virus-specific cytotoxic T lymphocytes (CTLs), we recently showed that polymorphonuclear neutrophils (PMNs) and the matrix-degrading metalloproteinases (MMPs) they produce are necessary for the intrahepatic recruitment of antigen nonspecific mononuclear cells that amplify the liver damage initiated by the CTLs. We now report that the high-mobility group box 1 protein (HMGB1) is also involved in this process. Transfer of CTLs in HBV transgenic mice induces the translocation of HMGB1 from the nucleus to the cytoplasm of hepatocytes surrounding CTL-containing necroinflammatory liver foci, without significant net synthesis of HMGB1. Treatment of CTL-injected HBV transgenic mice with either recombinant Box-A or glycyrrhizin, two functional inhibitors of extracellular HMGB1, significantly decreases the intrahepatic recruitment of PMNs and all other inflammatory cells, in the face of intact homing of virus-specific CTLs into the liver. The inhibition of PMN chemoattraction explains the mode of action of glycyrrhizin, which has long been used in Japan for the treatment of hepatitis, and suggests that new and more potent inhibitors of HMGB1 may be useful for the treatment of patients chronically infected with HBV.

Stimulation of net muscle protein synthesis by whey protein ingestion before and after exercise

Timing of nutrient ingestion has been demonstrated to influence the anabolic response of muscle following exercise. Previously, we demonstrated that net amino acid uptake was greater when free essential amino acids plus carbohydrates were ingested before resistance exercise rather than following exercise. However, it is unclear if ingestion of whole proteins before exercise would stimulate a superior response compared with following exercise. This study was designed to examine the response of muscle protein balance to ingestion of whey proteins both before and following resistance exercise. Healthy volunteers were randomly assigned to one of two groups. A solution of whey proteins was consumed either immediately before exercise (PRE; n = 8) or immediately following exercise (POST; n = 9). Each subject performed 10 sets of 8 repetitions of leg extension exercise. Phenylalanine concentrations were measured in femoral arteriovenous samples to determine balance across the leg. Arterial amino acid concentrations were elevated by approximately 50%, and net amino acid balance switched from negative to positive following ingestion of proteins at either time. Amino acid uptake was not significantly different between PRE and POST when calculated from the beginning of exercise (67 +/- 22 and 27 +/- 10 for PRE and POST, respectively) or from the ingestion of each drink (60 +/- 17 and 63 +/- 15 for PRE and POST, respectively). Thus the response of net muscle protein balance to timing of intact protein ingestion does not respond as does that of the combination of free amino acids and carbohydrate.

Volatile fatty acid proportions and microbial protein synthesis in the rumen of cattle receiving grass silage ensiled with different rates of formic acid

AbstractTimothy–meadow fescue herbage was ensiled with formic acid (FA) (expressed as 100% solution) at the rates of 0, 2, 4 or 6 L t−1. The silages were fed along with concentrates to bulls fitted with cannulae in the rumen and duodenum. The ration comprised grass silage (700 g kg−1), barley (240 g kg−1) and rapeseed meal (60 g kg−1). The application rate of FA had no effect on the site or extent of the digestion of dietary organic matter (OM) and neutral‐detergent fibre. The flow of total N at the duodenum increased linearly (P &lt; 0·05) with application rate of FA, reflecting mainly an increased (P &lt; 0·01) flow of microbial N. The apparent efficiency of net microbial protein synthesis in the rumen increased (P &lt; 0·05), the proportion of propionate in the volatile fatty acids (VFA) in the rumen was not affected (P &gt; 0·05) but that of butyrate increased (linear and quadratic effects, P &lt; 0·01) with increasing rate of FA. It is concluded that an increase in the rate of FA at ensiling leads to a higher utilization of energy and/or protein‐yielding substrates for rumen microbes and to a modified rumen VFA pattern with an increased proportion of butyrate.

Metabolic Response to Stress in the Neonate Who Has Surgery

After completing this article, readers should be able to: 1. Describe the basic metabolic pathways involved in neonatal nutrition. 2. Describe the effect of stress on nutrition and metabolism in the neonate undergoing surgery. 3. Describe the differences in the metabolic response to stress between an adult and a neonate. Over the past 4 decades, there have been remarkable improvements in our ability to care for critically ill infants. Progress in surgical and critical care of neonates has led to improved survival rates. Our understanding of the nutritional requirements of infants continues to advance. In contrast to years past, practitioners caring for critically ill infants undergoing surgery today have various nutritional options, including specialized parenteral and enteral formulations. Although our understanding of host defense and nutritional support continues to evolve, the interplay between the immune system and metabolism is being investigated. Hence, we are improving the match between the infant’s energy expenditure and nutritional requirements. Critical to further progress is additional understanding of the metabolic response of a neonate to surgical stress. All living organisms require energy to survive. Fuel sources enter into complex metabolic cascades to form the ultimate energy substrate, adenosine triphosphate (ATP), a fuel needed to drive and maintain all physiologic systems. Under stressful conditions, intrinsic substrate mobilizes to synthesize additional energy substrate. In essence, host substrate is mobilized for the promotion of healing after injury. Cytokines, released in response to injury, drive the catabolic response to stress, but in abundance, as with the systemic inflammatory response syndrome (SIRS), cytokines are detrimental to the organism. The nutritional requirements of a neonate are significantly greater than those of an adult because not only are there requirements for substrate intake to fulfill basic metabolic needs, but there are metabolic needs to maintain rapid and continued growth and development. Furthermore, the metabolic response …

Oestradiol Up‐Regulates Glutamine Synthetase mRNA and Protein Expression in the Hypothalamus and Hippocampus: Implications for a Role of Hormonally Responsive Glia in Amino Acid Neurotransmission

Rapidly emerging evidence suggests that glial cells in the central nervous system are sensitive to oestrogen actions. However, the functional consequences of the cellular mechanisms of these cells have proven difficult to study in vivo because of the intimate relationships between neurones and glia. Microarray technology offers the potential to uncover steroid hormone regulation of glial-specific genes that may play a role in hormone-dependent neuronal-glial interactions. Analysis of transcriptomes from the medial basal hypothalamus (MBH) of oestradiol and vehicle-treated adult ovariectomised mice revealed an up-regulation of several glial specific genes by oestradiol, including glutamine synthetase (GS), which facilitates the conversion of glutamate to glutamine and plays an integral role in amino acid neurotransmission. In situ hybridisation confirmed that oestradiol treatment resulted in an up-regulation of GS gene expression in the arcuate and ventromedial nuclei of the MBH, as well as the medial amygdala and hippocampus. Moreover, oestradiol increased protein expression of GS in both the MBH and hippocampus. Neurones are incapable of de novo net synthesis of glutamate from glucose and are dependent on glial-provided precursors such as glutamine to renew their amino acid transmitter pools. Thus, oestradiol induced expression of GS suggests a significant role for glial cells in hormonal modulation of glutamatergic neurotransmission important to female reproductive behaviours, neuroendocrine physiology and cognitive functions.

Serum ethanolamine and hepatocyte proliferation in perinatal and partially hepatectomized rats

It has been shown that the administration of ethanolamine (Etn) to partially hepatectomized rats enhances stimulation of DNA synthesis in regenerating hepatocytes. The present study aimed to test the hypothesis that the level of serum Etn in vivo may be regulated to control the growth of hepatocytes. Concentrations of serum Etn were determined in rats 1) of varying ages (from embryonic-19 (E-19) to 7-week-old), and 2) during regeneration following two-thirds hepatectomy (PH), to investigate whether serum Etn concentration correlates with the rate of proliferation of hepatocytes in growing animals or during regeneration. Serum Etn levels were 3 fold higher in E-19 fetuses and newborns than in adults, and were increased 2 fold 4 h after PH and remained high for at least 24 h. Results in both systems indicated a significant positive correlation between the rate of hepatocyte proliferation and serum Etn levels. Furthermore, Etn supplementation of 0.1 to 1 mmol immediately after PH promoted a significant weight gain and stimulated phosphatidylethanolamine (PE) and phosphatidylcholine (PC) synthesis in the regenerating liver. We also observed that whenever serum Etn levels were elevated, the metabolism of PE and PC in the liver changed dynamically, first by elevating the net synthesis of PE. Taken together, these results suggested that the levels of serum Etn might be regulated based on the physiological state of an animal, which consequently regulates the proliferation of hepatocytes.

The Effect of Source of Dietary Fiber and Starch on Ileal and Fecal Amino Acid Digestibility in Growing Pigs

Studies were carried out with a repeated 4x4 Latin square design with eight cannulated pigs fed four experimental diets to investigate the effect of dietary fiber and starch sources on apparent ileal and fecal amino acid digestibility. Each period lasted 15 d, with diet acclimation from d 1 to 7, feces collection for 48 h on d 8 to 9 and ileal sample collection for 12 h on d 13 to 15. The four experimental diets consisted mainly of cooked rice with the addition of protein sources (CON), partial replacement of cooked rice with either potato starch (PS), sugar beet pulp (SBP) or wheat bran (WB). Chromic oxide was used as an indigestible marker. With the exception of histidine, lysine and tryptophan, no differences were observed in the apparent ileal digestibility of amino acids between diets. The inclusion of potato starch did not affect the ileal and fecal amino acid digestibility. In comparison with diet CON, a decreased (p<0.05) ileal digestibility of histidine was found in pigs fed diet SBP, while the ileal digestibilities of histidine, lysine and tryptophan were decreased (p<0.05) by the inclusion of wheat bran. Inclusion of fiber sources (sugar beet pulp and wheat bran) caused a reduction (p<0.05) in the fecal amino acid digestibility and the net disappearance of amino acids in the large intestine. Of the indispensable amino acids, there was a 'net synthesis' for methionine in the large intestine of pigs when diets were supplemented with dietary fiber. The decrease in fecal amino acid digestibility with the addition of dietary fiber indicates an increase in the synthesis of bacterial protein in the large intestine.

Effects of (−)-hydroxycitrate on net fat synthesis as de novo lipogenesis

(−)-Hydroxycitrate (HCA) might promote weight maintenance by limiting the capacity for de novo lipogenesis (DNL). It was investigated whether HCA may reduce DNL in humans during a persistent excess of energy intake as carbohydrate. In a double-blind, placebo-controlled, randomized and cross-over design, 10 sedentary lean male subjects (mean ± S.D., age: 24 ± 5 years, BMI: 21.8 ± 2.1 kg/m 2) performed a glycogen depletion exercise test followed by a 3-day high-fat diet (F/CHO/P, 60/25/15% energy; 100% of energy expenditure (EE)) and a 7-day high-CHO diet (F/CHO/P, < 5/> 85/10% energy; 130–175% of EE; overfeeding). During overfeeding, they ingested 3 × 500 mg/day HCA or placebo (PLA). Each intervention ended with a 60-h stay in the respiration chamber (days 9 and 10). Body weight increased during overfeeding (mean ± S.E., HCA: 2.9 ± 0.2 kg, PLA: 2.8 ± 0.2 kg). Respiratory quotient (RQ) was > 1.00 in all subjects indicating that DNL was present. On day 9, 24-h EE was lower with HCA compared to PLA ( P < 0.05). On day 10, resting metabolic rate and RQ during night were lower ( P < 0.01 and P < 0.05, respectively). Non-protein RQ, fat balance and net fat synthesis as DNL tended to be lower ( P < 0.1) with HCA compared to PLA indicating lower DNL; activity-induced EE was higher with HCA ( P < 0.05) indicating the urge to eliminate the excess of energy ingested. We conclude that an experimental condition resulting in DNL in humans was created and that treatment with HCA during overfeeding with carbohydrates may reduce DNL.

Mutagenic Definition of a Papain-Like Catalytic Triad, Sufficiency of the N-Terminal Domain for Single-Site Core Catalytic Enzyme Acylation, and C-Terminal Domain for Augmentative Metal Activation of a Eukaryotic Phytochelatin Synthase

Phytochelatin (PC) synthases are gamma-glutamylcysteine (gamma-Glu-Cys) dipeptidyl transpeptidases that catalyze the synthesis of heavy metal-binding PCs, (gamma-Glu-Cys)nGly polymers, from glutathione (GSH) and/or shorter chain PCs. Here it is shown through investigations of the enzyme from Arabidopsis (Arabidopsis thaliana; AtPCS1) that, although the N-terminal half of the protein, alone, is sufficient for core catalysis through the formation of a single-site enzyme acyl intermediate, it is not sufficient for acylation at a second site and augmentative stimulation by free Cd2+. A purified N-terminally hexahistidinyl-tagged AtPCS1 truncate containing only the first 221 N-terminal amino acid residues of the enzyme (HIS-AtPCS1_221tr) is competent in the synthesis of PCs from GSH in media containing Cd2+ or the synthesis of S-methyl-PCs from S-methylglutathione in media devoid of heavy metal ions. However, whereas its full-length hexahistidinyl-tagged equivalent, HIS-AtPCS1, undergoes gamma-Glu-Cys acylation at two sites during the Cd2+-dependent synthesis of PCs from GSH and is stimulated by free Cd2+ when synthesizing S-methyl-PCs from S-methylglutathione, HIS-AtPCS1_221tr undergoes gamma-Glu-Cys acylation at only one site when GSH is the substrate and is not directly stimulated, but instead inhibited, by free Cd2+ when S-methylglutathione is the substrate. Through the application of sequence search algorithms capable of detecting distant homologies, work we reported briefly before but not in its entirety, it has been determined that the N-terminal half of AtPCS1 and its equivalents from other sources have the hallmarks of a papain-like, Clan CA Cys protease. Whereas the fold assignment deduced from these analyses, which substantiates and is substantiated by the recent determination of the crystal structure of a distant prokaryotic PC synthase homolog from the cyanobacterium Nostoc, is capable of explaining the strict requirement for a conserved Cys residue, Cys-56 in the case of AtPCS1, for formation of the biosynthetically competent gamma-Glu-Cys enzyme acyl intermediate, the primary data from experiments directed at determining whether the other two residues, His-162 and Asp-180 of the putative papain-like catalytic triad of AtPCS1, are essential for catalysis have yet to be presented. This shortfall in our basic understanding of AtPCS1 is addressed here by the results of systematic site-directed mutagenesis studies that demonstrate that not only Cys-56 but also His-162 and Asp-180 are indeed required for net PC synthesis. It is therefore established experimentally that AtPCS1 and, by implication, other eukaryotic PC synthases are papain Cys protease superfamily members but ones, unlike their prokaryotic counterparts, which, in addition to having a papain-like N-terminal catalytic domain that undergoes primary gamma-Glu-Cys acylation, contain an auxiliary metal-sensing C-terminal domain that undergoes secondary gamma-Glu-Cys acylation.

Biotransformation of mycosporine like amino acids (MAAs) in the toxic dinoflagellate Alexandrium tamarense

Changes in mycosporine-like amino acids (MAAs) induced by the increase of photosynthetically active radiation (PAR) were studied in the toxic dinoflagellate Alexandrium tamarense. Cultures of A. tamarense were maintained at exponential growth under low (25 μmol quanta m −2 s −1) PAR irradiance. The cultures were nutrient enriched and one day later exposed to higher irradiance (150 μmol quanta m −2 s −1). The content of MAAs was determined by means of high performance liquid chromatography (HPLC). Eleven MAAs, including some partially characterized compounds, were identified. The MAAs synthesis induction can be described as a two-stage process. The first one involves the net synthesis of the MAAs bi-substituted by amino acids. In the second stage these compounds were transformed into other secondary MAAs. The two most prominent changes were observed in the concentration of porphyra-334 and palythene. The cellular concentration of porphyra-334 increased during the first 2 h of exposure to higher irradiance and then decreased rapidly. In contrast, the cellular concentration of palythene showed a continuous accumulation since the beginning of the exposure. In A. tamarense the main route of MAAs transformation has porphyra-334 as a precursor of a sequential conversion resulting in the accumulation of palythene.

Effect of Total Mixed Ration Composition on Fermentation and Efficiency of Ruminal Microbial Crude Protein Synthesis In Vitro

The goal of this study was to identify dietary factors that affect fermentation and efficiency of microbial crude protein (CPM) synthesis in the rumen in vitro. We used 16 total mixed, dairy cow rations with known digestibilities that varied in ingredient composition and nutrient content. Each ration was incubated in a Rusitec (n = 3) for 15 d, and fermentation of different fractions was assessed. Observed extents of fermentation in 24h were 35 to 47% for organic matter, 25 to 60% for crude protein, 3 to 28% for neutral detergent fiber, and 31 to 45% for gross energy. Organic matter fermentation depended on the content of crude protein and neutral detergent fiber in the ration. We studied net synthesis of CPM using an 15N dilution technique and found that 7 d of continuous 15N application are needed to achieve an 15N enrichment plateau in the N of isolated microbes in this type of study. The efficiency of CPM synthesis was 141 to 286 g/kg of fermented organic matter or 4.9 to 11.1 g/MJ of metabolizable energy, and these ranges agree with those found in the literature. Multiple regressions to predict the efficiency of CPM synthesis by diet data showed that crude protein was the only dietary chemical fraction that had a significant effect. Fat content and the inclusion rate of corn silage in the ration also tended to improve efficiency. We suggest that microbial need for preformed amino acids may explain the crude protein effect. A large part of the variation in efficiency of microbial activity still remains unexplained.

Glial Modulation of GABAergic and Glutamat ergic Neurotransmission

Function of astroglia in the modulation of availability, release and clearance (inactivation) of Glu and GABA within the central nervous system is reviewed. Net synthesis of Glu through Gln synthetase exclusively localized in astrocytes can only occur by a metabolic coupling between neurons and astrocytes. Two (GLAST and GLT-1) of the five Glu transporters cloned preferentially expressed in astrocytes perform the astroglial Glu uptake of very high capacity. Moreover, astrocytes have been shown to mediate Glu release by a mechanism mimicking vesicular release. Biosynthesis of GABA in neurons is brought about by decarboxylation of Glu catalyzed by a pyridoxal phosphate requiring enzyme (GAD) that exists in two isoforms (GAD65 and GAD67) exhibiting different subcellular localization and regulatory properties. Detailed studies of GABA synthesis in GABAergic neurons using (13)C NMR spectroscopy have provided evidence for direct involvement of the tricarboxylic acid cycle. Synaptically released GABA taken up into surrounding astrocytes is converted to either CO(2) or Gln. Two reports on the release of GABA in rat dorsal root ganglia indicated that glial cells may perform GABA release as well. Gln formed from GABA in astrocytes can be transferred to GABAergic neurons and subsequently converted to GABA. Inhibition of either degradation or clearance of GABA has been successfully applied in the development of antiepileptics such as vigabatrin or tiagabine. So far, no specific strategy has been developed aimed at stimulating Glu transport.

Milk Ingestion Stimulates Net Muscle Protein Synthesis following Resistance Exercise

Previous studies have examined the response of muscle protein to resistance exercise and nutrient ingestion. Net muscle protein synthesis results from the combination of resistance exercise and amino acid intake. No study has examined the response of muscle protein to ingestion of protein in the context of a food. This study was designed to determine the response of net muscle protein balance following resistance exercise to ingestion of nutrients as components of milk. Three groups of volunteers ingested one of three milk drinks each: 237 g of fat-free milk (FM), 237 g of whole milk (WM), and 393 g of fat-free milk isocaloric with the WM (IM). Milk was ingested 1 h following a leg resistance exercise routine. Net muscle protein balance was determined by measuring amino acid balance across the leg. Arterial concentrations of representative amino acids increased in response to milk ingestion. Threonine balance and phenylalanine balance were both > 0 following milk ingestion. Net amino acid uptake for threonine was 2.8-fold greater (P < 0.05) for WM than for FM. Mean uptake of phenylalanine was 80 and 85% greater for WM and IM, respectively, than for FM, but not statistically different. Threonine uptake relative to ingested was significantly (P < 0.05) higher for WM (21 +/- 6%) than FM (11 +/- 5%), but not IM (12 +/- 3%). Mean phenylalanine uptake/ingested also was greatest for WM, but not significantly. Ingestion of milk following resistance exercise results in phenylalanine and threonine uptake, representative of net muscle protein synthesis. These results suggest that whole milk may have increased utilization of available amino acids for protein synthesis.