Important impacts of intestinal bacteria on utilization of dietary amino acids in pigs

Bacteria in pig intestine can actively metabolize amino acids (AA). However, little research has focused on the variation in AA metabolism by bacteria from different niches. This study compared the metabolism of AA by microorganisms derived from the lumen and epithelial wall of the pig small intestine, aiming to test the hypothesis that the metabolic profile of AA by gut microbes was niche specific. Samples from the digesta, gut wall washes and gut wall of the jejunum and ileum were used as inocula. Anaerobic media containing single AA were used and cultured for 24 h. The 24-h culture served as inocula for the subsequent 30 times of subcultures. Results showed that for the luminal bacteria, all AA concentrations except phenylalanine in the ileum decreased during the 24-h in vitro incubation with a increase of ammonia concentration, while 4 AA (glutamate, glutamine, arginine and lysine) in the jejunum decreased, with the disappearance rate at 60–95 %. For tightly attached bacteria, all AA concentrations were generally increased during the first 12 h and then decreased coupled with first a decrease and then an increase of ammonia concentration, suggesting a synthesis first and then a catabolism pattern. Among them, glutamate in both segments, histidine in the jejunum and lysine in the ileum increased significantly during the first 12 h and then decreased at 24 h. The concentrations of glutamine and arginine did not change during the first 12 h, but significantly decreased at 24 h. Jejunal lysine and ileal threonine were increased for the first 6 or 12 h. For the loosely attached bacteria, there was no clear pattern for the entire AA metabolism. However, glutamate, methionine and lysine in the jejunum decreased after 24 h of cultivation, while glutamine and threonine in the jejunum and glutamine and lysine in the ileum increased in the first 12 h. During subculture, AA metabolism, either utilization or synthesis, was generally decreased with disappearance rate around 20–40 % for most of AA and negligible for branch chained AA (BCAA). However, the disappearance rate of lysine in each group was around 90 % throughout the subculture, suggesting a high utilization of lysine by bacteria from all three compartments. Analysis of the microbial community during the 24-h in vitro cultivation revealed that bacteria composition in most AA cultures varied between different niches (lumen and wall-adherent fractions) in the jejunum, while being relatively similar in the ileum. However, for isoleucine and leucine cultures, bacteria diversity was similar between the luminal fraction and tightly attached fraction, but significantly higher than in the loosely attached fraction. For glutamine and valine cultures, bacteria diversity was similar between the luminal and loosely attached fractions, but lower than that of tightly attached bacteria. After 30 subcultures, bacteria diversity in arginine, valine, glutamine, and leucine cultures varied between niches in the jejunum while being relatively stable in the ileum, consistent with those in the 24-h in vitro cultures. The findings may suggest that luminal bacteria tended to utilize free AA, while tightly attached adherent bacteria seemed in favor of AA synthesis, and that small intestinal microbes contributed little to BCAA metabolism.     

Response of HEK293 and CHO cells overexpressing fusiogenic syncytin-1 to mitochondrion-mediated apoptosis induced by antimycin A

Apoptosis is essential for the regulation of cellular homeostasis in the placenta and is also involved in the pathophysiology of pregnancy-related diseases such as pre-eclampsia and intrauterine growth restriction (IUGR). Syncytin-1, a fusiogenic glycoprotein of endogenous-retroviral origin expressed in human trophoblasts, facilitates placental syncytium formation and is found reduced in pre-eclamptic placentas. We focus here on the mitochondrial apoptotic pathway and investigate whether the overexpression of syncytin-1 in HEK293-52 (human embryonic kidney cells) and CHO-52 cells influences the apoptotic response to the mitochondrial inhibitor antimycin A (AA). After the induction of apoptosis by 5 microM AA and incubation for up to 36 h in the absence of serum, the mean apoptotic rate was reduced by 15-30% in syncytin-1 transfected cells compared with mock-transfectants. After 12 h of challenge with AA we found lower cytochrome c levels in the cytoplasmic protein fraction and higher amounts in the mitochondrial fraction in syncytin-1 transfectants compared with mock-transfectants. We observed a decreased Mitotracker Red staining of mitochondria following AA challenge for 24 h in mock-treated CHO cells, in particular, compared with syncytin-1 transfectants. Moreover, we found a reduced activation of caspase 9 in syncytin-1 transfected HEK293-52 cells after 48 h of apoptotic challenge compared to mock-transfectants. However, a high expression of anti-apoptotic Bcl-x(L) was found in both cell types. Using syncytin-1 transfected HEK293-52 cells and CHO-52 cells, we provide initial evidence that syncytin-1 may exert its anti-apoptotic function at the mitochondrial level. A reduced release of cytochrome c followed by a diminished activation of caspase 9 is a possible mechanism.     

Rapid remodeling of arachidonate from phosphatidylcholine to phosphatidylethanolamine pools during mast cell activation.

The objective of the present study was to better understand the remodeling of arachidonic acid (AA) in phospholipids of the mouse bone marrow-derived mast cell (BMMC) during Ag and ionophore A23187 activation. Initial studies were designed to understand the movement of AA in phospholipid classes under resting conditions. BMMC pulse labeled with AA incorporated greater than 95% of the label into the major phospholipid classes. Phosphatidylcholine (PC) subclasses, 1-acyl-2-arachidonoyl-(sn-glycero-3-phosphocholine (GPC)) in particular, initially accounted for most of the label incorporated into the cells with phosphatidylinositol/phosphatidylserine (PI/PS) and phosphatidylethanolamine (PE) subclasses containing much smaller quantities. Prolonged incubation of labeled BMMC resulted in a decrease in the radioactivity in PC with a concomitant increase in PE such that 1-alk-1-enyl-2-arachidonoyl-(sn-glycero-3-phosphoethanolamine (GPE)) became the single largest labeled AA pool by 24 h. Further experiments indicated that 24 h was the time required to reach isotopic equilibrium among AA-containing phospholipids of the BMMC. In the next series of experiments, BMMC phospholipids were labeled to different specific activities by either labeling the cells for 0.5 h or for 24 h followed by stimulation. Under isotopic equilibrium conditions (24 h), stimulation resulted in AA release from PE greater than PC much greater than PI/PS with 1-alk-1-enyl-2-arachidonoyl-GPE providing the bulk of AA released from the BMMC. By contrast, cells labeled for 0.5 h released AA from PC much greater than PI/PS, with 1-acyl-2-arachidonoyl-GPC accounting for most of the AA released from BMMC phospholipids. Label associated with PE subclasses under nonequilibrium conditions remained unchanged or slightly increased throughout a 10-min stimulation period. Finally, BMMC were double labeled with [14C]-AA for 24 h and then with [3H]-AA for 0.5 h. Cell stimulation resulted in a decrease in the [3H]/[14C] ratio in PC and PI and an increase in the ratio in PE. The decrease in [3H]/[14C] ratio in PC was mainly in 1-acyl-2-arachidonoyl-GPC, whereas the increase in PE subclasses was primarily in 1-alk-1-enyl-2-arachidonoyl-GPE. The [3H]/[14C] ratio in cellular neutral lipids and in supernatant fluid products were at values between PC and PE subclasses. Taken together, these data suggest that during Ag activation, the release of free arachidonic acid is from predominantly PE subclasses. Concomitant with the release of AA, there is a rapid remodeling of AA from PC subclasses into PE subclasses (1-alk-1-enyl-2-acyl-GPE).     

Preferential distribution of long chain polyunsaturated fatty acids in phospatidyl ethanolamine fraction of guinea pig alveolar apical membranes

We investigated the fatty acid distribution in guinea pig alveolar apical membranes at different developmental stages. Fatty acid composition of the purified membranes isolated from guinea pig fetuses (at 65 day, term=68 day), neonates (day 1) and adult males was determined. The levels of arachidonic acid (AA) and docosahexaenoic acid (DHA) were higher in the adult guinea pig alveolar apical membrane phosphatidylethanolamine (PE) fraction (9. 3+/-2.2 and 2.9+/-1.0%, respectively) while in other phospholipids (PL) fractions their levels were low or absent (P<0.01). Furthermore, levels of AA and DHA in the PE fraction of apical membrane increased significantly from fetal (6.6+/-3.0 and 0.8+/-0.4%, respectively) to neonatal life (10.3+/-1.5 and 3.0+/-0.8%, respectively). Increase in the level of DHA (almost four-fold) was much more pronounced than that of AA (P<0.05). As for guinea pig alveolar membranes, EPA and AA were mostly present in the PE fraction in pulmonary adenocarcinoma derived cells (A549 cells), a parallel model of type II pneumocytes, with the levels of AA around three-fold greater than that of EPA, Binding of radiolabelled fatty acids to A549 cells showed no significant differences between the maximum uptake achieved for different fatty acids (AA, 1.7+/-0.2, EPA, 2.3+/-0.3, LA, 1.7+/-0.2, OA, 2.0+/-0.2nmol/mg protein, P>0.5). Once the fatty acids were taken up by these cells AA was mostly identifiable in the monoacylglycerol (MAG) fraction, whereas EPA was equally distributed between the MAG and PL fractions. Oleic acid was mainly present in the triglyceride (TAG) fraction whereas LA was evenly distributed between the TAG, MAG, and PL fractions. Our data demonstrate a preferential distribution of AA and DHA in PE fractions of alveolar apical membranes during development.     

Rapid stimulation of 5-lipoxygenase activity in potato by the fungal elicitor arachidonic Acid

The activity of lipoxygenase (LOX) in aged potato tuber discs increased by almost 2-fold following treatment of the discs with the fungal elicitor arachidonic acid (AA). Enzyme activity increased above that in untreated discs within 30 min after AA treatment, peaked at 1 to 3 h, and returned to near control levels by 6 h. The majority of the activity was detected in a soluble fraction (105,000g supernatant), but a minor portion was also associated with a particulate fraction enriched in microsomal membranes (105,000g pellet); both activities were similarly induced. 5-Hydroperoxyeicosatetraenoic acid was the principal product following incubation of these extracts with AA. Antibodies to soybean LOX strongly reacted with a protein with a molecular mass of approximately 95-kD present in both soluble and particulate fractions whose abundance generally corresponded with LOX activity in extracts. LOX activity was not enhanced by treatment of the discs with nonelicitor fatty acids or by branched β-glucans from the mycelium of Phytophthora infestans. Prior treatment of the discs with abscisic acid, salicylhydroxamic acid, or n-propyl gallate, all of which have been shown to suppress AA induction of the hypersensitive response, inhibited the AA-induced increment in LOX activity. Cycloheximide pretreatment, which abolishes AA elicitor activity for other responses such as phytoalexin induction, did not inhibit LOX activity in water- or elicitor-treated discs but enhanced activity similar to that observed by AA treatment. The elicitor-induced increase in 5-LOX activity preceded or temporally paralleled the induction of other studied responses to AA, including the accumulation of mRNAs for 3-hydroxy-3-methylglutaryl coenzyme A reductase and phenylalanine ammonia lyase reported here. The results are discussed in relation to the proposed role of the 5-LOX in signal-response coupling of arachidonate elicitation of the hypersensitive response.     

Amino acid repletion does not decrease muscle protein catabolism during hemodialysis

Intradialytic protein catabolism is attributed to loss of amino acids in the dialysate. We investigated the effect of amino acid infusion during hemodialysis (HD) on muscle protein turnover and amino acid transport kinetics by using stable isotopes of phenylalanine, leucine, and lysine in eight patients with end-stage renal disease (ESRD). Subjects were studied at baseline (pre-HD), 2 h of HD without amino acid infusion (HD-O), and 2 h of HD with amino acid infusion (HD+AA). Amino acid depletion during HD-O augmented the outward transport of amino acids from muscle into the vein. Increased delivery of amino acids to the leg during HD+AA facilitated the transport of amino acids from the artery into the intracellular compartment. Increase in muscle protein breakdown was more than the increase in synthesis during HD-O (46.7 vs. 22.3%, P < 0.001). Net balance (nmol.min(-1).100 ml (-1)) was more negative during HD-O compared with pre-HD (-33.7 +/- 1.5 vs. -6.0 +/- 2.3, P < 0.001). Despite an abundant supply of amino acids, the net balance (-16.9 +/- 1.8) did not switch from net release to net uptake. HD+AA induced a proportional increase in muscle protein synthesis and catabolism. Branched chain amino acid catabolism increased significantly from baseline during HD-O and did not decrease during HD+AA. Protein synthesis efficiency, the fraction of amino acid in the intracellular pool that is utilized for muscle protein synthesis decreased from 42.1% pre-HD to 33.7 and 32.6% during HD-O and HD+AA, respectively (P < 0.01). Thus amino acid repletion during HD increased muscle protein synthesis but did not decrease muscle protein breakdown.     

Biodegradation of a crude oil by three microbial consortia of different origins and metabolic capabilities

Microbial consortia were obtained three by sequential enrichment using different oil products. Consortium F1AA was obtained on a heavily saturated fraction of a degraded crude oil; consortium TD, by enrichment on diesel and consortium AM, on a mixture of five polycyclic aromatic hydrocarbons [PAHs]. The three consortia were incubated with a crude oil in order to elucidate their metabolic capabilities and to investigate possible differences in the biodegradation of these complex hydrocarbon mixtures in relation to their origin. The efficiency of the three consortia in removing the saturated fraction was 60% (F1AA), 48% (TD) and 34% (AM), depending on the carbon sources used in the enrichment procedures. Consortia F1AA and TD removed 100% of n-alkanes and branched alkanes, whereas with consortium AM, 91% of branched alkanes remained. Efficiency on the polyaromatic fraction was 19% (AM), 11% (TD) and 7% (F1AA). The increase in aromaticity of the polyaromatic fraction during degradation of the crude oil by consortium F1AA suggested that this consortium metabolized the aromatic compounds primarily by oxidation of the alkylic chains. The 500-fold amplification of the inocula from the consortia by subculturing in rich media, necessary for use of the consortia in bioremediation experiments, showed no significant decrease in their degradation capability. Journal of Industrial Microbiology & Biotechnology (2002) 28, 252–260 DOI: 10.1038/sj/jim/7000236 Received 12 July 2001/ Accepted in revised form 11 November 2001     

Protective effects of ascorbic acid on arterial hemodynamics during acute hyperglycemia

Mortality increases when acute coronary syndromes are complicated by stress-induced hyperglycemia. Early pulse wave reflection can augment central aortic systolic blood pressure and increase left ventricular strain. Altered pulse wave reflection may contribute to the increase in cardiac risk during acute hyperglycemia. Chronic ascorbic acid (AA) supplementation has recently been shown to reduce pulse wave reflection in diabetes. We investigated the in vivo effects of acute hyperglycemia, with and without AA pretreatment, on pulse wave reflection and arterial hemodynamics. Healthy male volunteers were studied. Peripheral blood pressure (BP) was measured at the brachial artery, and the SphygmoCor pulse wave analysis system was used to derive central BP, the aortic augmentation index (AIx; measure of systemic arterial stiffness), and the time to pulse wave refection (Tr; measure of aortic distensibility) from noninvasively obtained radial artery pulse pressure (PP) waveforms. Hemodynamics were recorded at baseline and then every 30 min during a 120-min systemic hyperglycemic clamp (14 mmol/l). The subjects, studied on two separate occasions, were randomized in a double-blind, crossover manner to placebo or 2 g intravenous AA before the initiation of hyperglycemia. During hyperglycemia, AIx increased and Tr decreased. Hyperglycemia did not change peripheral PP but did magnify central aortic PP and diminished the normal physiological amplification of PP from the aorta to the periphery. Pulse wave reflection, as assessed from peripheral pulse wave analysis, is enhanced during acute hyperglycemia. Pretreatment with AA prevented the hyperglycemia-induced hemodynamic changes. By protecting hemodynamics during acute hyperglycemia, AA may have therapeutic use.     

Prevalence and Clinical Characteristics Associated with Pulmonary Hypertension in African-Americans

Background

Pulmonary hypertension (PH) is associated with increased mortality and morbidity. It is frequently associated with cardiopulmonary diseases that are prevalent in African Americans (AAs). However, the prevalence or determinants of PH in the AA population is not known.

Methods

We conducted a cross-sectional study to estimate the prevalence of PH (defined as trans-tricuspid gradient ≥ 35 mm Hg) and associated clinical characteristics in AAs using the Jackson Heart Study cohort (n=3,282) who underwent echocardiography and had a measurable trans-tricuspid regurgitant jet. Echocardiography is frequently used for screening for PH despite its limitations in estimating accurate PA systolic pressures. Overall and age-adjusted gender-specific prevalence were estimated and modified Poisson regression was used to identify independent clinical, spirometric, and echocardiographic characteristics associated with PH.

Results

The mean age of the study population was 56.1 ± 12.6 years with 67.5% female. The prevalence of PH was 6.8%, with higher prevalence in female AAs (age-adjusted prevalence: Men 4.9%, 95% CI 3.6-6.2%; Women 7.7%, 95% CI 6.6-8.8%). Pulmonary hypertension prevalence increased with age (Prevalence Ratio: 10.0, 95%CI 4.0-25.1, >65 versus <45 years old), presence of obesity, higher pulse pressure, diabetes, obstructive or restrictive spirometry pattern, and severe left heart valvular disease. Also, PH was significantly associated with left atrial size and left ventricular ejection fraction.

Conclusions

Pulmonary hypertension is prevalent in AAs, more in women than in men. The identified cardiopulmonary risk factors that increase the prevalence of PH may assist in diagnosis and management of these at-risk subjects in the AA population.     

Daily profiles of plasma prolactin (PRL), growth hormone (GH), insulin-like growth factor-1 (IGF-1), luteinizing hormone (LH), testosterone, and melatonin, and of pituitary PRL mRNA and GH mRNA in male Long Evans rats in acute phase of adjuvant arthritis

We studied the effects of adjuvant arthritis (AA) on the endocrine circadian rhythms of plasma prolactin (PRL), growth hormone (GH), insulin-like growth factor-1 (IGF-1), luteinizing hormone (LH), testosterone, and melatonin and of pituitary PRL and GH mRNA in male Long Evans rats. Groups of control and AA rats (studied 23 days after AA induction) that were housed under a 12/12 h light/dark cycle (light on at 06:00 h) were killed at 4 h intervals starting at 14:00 h. Cosinor analysis revealed a significant 12 h rhythm in PRL and PRL mRNA (p < 0.001) in controls with peaks at 14:00 h and 02:00 h, respectively. The peak at 02:00 h was abolished in the AA group resulting in a significant 24 h rhythm in parallel with that of PRL (p < 0.05) and PRL mRNA (p < 0.0001). Growth hormone showed no rhythm, but a significant rhythm of GH mRNA was present in both groups (p < 0.0001). Insulin-like growth factor-1 showed a 24 h rhythm in control but not in AA rats. The mean values of GH, GH mRNA, and IGF-1 were significantly reduced in AA. Luteinizing hormone displayed a significant 24 h rhythm (p < 0.01) peaking in the dark period in the control but not AA group. Testosterone showed in phase temporal changes of LH levels with AA abolishing the 02:00 h peak. Melatonin exhibited a significant 24 h rhythm in control (p < 0.001) and AA (p < 0.01) rats with maximum levels during the dark phase; the mesor value was higher in the AA males. These results demonstrate that AA interferes with the rhythms of all the studied hormones except the non-24 h (arrhythmic) GH secretion pattern and the rhythm in melatonin. The persistence of a distinct melatonin rhythm in AA suggests the observed disturbances of hormonal rhythms in this condition do not occur at the level of the pineal gland.     

Synthesis,subcellular distribution and turnover of dopamine beta-hydroxylase in organ cultures of sympathetic ganglia and adrenal medullae

The synthesis, subcellular distribution and turnover of dopamine beta-hydroxylase was studied in organ cultures of rat adrenal medullae and superior cervical ganglia. After exposure to [3H]leucine for 1 or 3 h, the tissues were homogenized at various time intervals and the amount of labelled dopamine beta-hydroxylase in different subcellular fractions (cytosol, soluble and membrane-bound fraction of catecholamine storage vesicles) was determined by immunoprecipitation and subsequent electrophoresis. In cultured adrenal medullae, induction of dopamine beta-hydroxylase initiated in vivo by administration of reserpine affected both soluble and membrane-bound pools of dopamine beta-hydroxylase to a similar extent after pulse-labelling for 1 or 3 h. The half-lives of dopamine beta-hydroxylase, which amounted to 6 h for the cytosol, 7.5 h for the soluble vesicular and 32 h for the membrane-bound vesicular pools were not altered by pretreatment with reserpine. In superior cervical ganglia the half-lives of the soluble pools were 2-3 times longer than in the adrenal medulla, whereas the half-life of the membrane-bound fraction was the same as in the adrenal medulla. In both organs the most heavily labelled fraction (both after a pulse of 1 or 3 h) was always that of the vesicular membrane, suggesting that newly-synthesized dopamine beta-hydroxylase is immediately incorporated into the storage vesicles and not via release into the cytosol from the site of synthesis. The fact that the half-life of membrane-bound dopamine beta-hydroxylase is markedly longer than that of the two soluble pools suggests that the single pools are not only independently supplied by newly-synthesized DBH but there is also no appreciable subsequent exchange between soluble and membrane-bound pools.     

Exogenous and endogenous contributions to nitrogen fluxes in the digestive tract of pigs fed a casein diet. II. Ileal and faecal digestibilities and absorption of amino acids.

The present work aimed at quantifying nitrogen (N) and amino acid (AA) fluxes in the digestive tract of growing pigs fed a casein diet. In this paper we report on digesta passage at the terminal ileum, on apparent balances at the ileal and faecal levels, and on nutrients appearance in the portal vein. Digesta flow-rate at the terminal ileum was maximum between 6 and 12 h after the meal. About 10% of N and 5% of total AA ingested were recovered within 24 h. AA absorption started 30 min after the meal, and was measurable until 13 to 14 h. The total AA absorbed in 24 h accounted for 128% of the AA ingested. The AA composition of ileal digesta was very different from that of casein, closely resembling that of endogenous proteins. The AA composition of faeces was very close to that of bacterial proteins. The ileal digestibilities of AA, though lower than their faecal values, were very high. This was confirmed by AA absorption balances greater than 100%. These data suggest that casein was almost totally digested by the terminal ileum, and that endogenous AA were substantially reabsorbed. These findings are supported by data on endogenous N recycling (15N), reported in a following paper.     

Role of Ascorbate in Detoxifying Ozone in the Apoplast of Spinach (Spinacia oleracea L.) Leaves

Both reduced and oxidized ascorbate (AA and DHA) are present in the aqueous phase of the extracellular space, the apoplast, of spinach (Spinacia oleracea L.) leaves. Fumigation with 0.3 [mu]L L-1 of ozone resulted in ozone uptake by the leaves close to 0.9 pmol cm-2 of leaf surface area s-1. Apoplastic AA was slowly oxidized by ozone. The initial decrease of apoplastic AA was <0.1 pmol cm-2 s-1. The apoplastic ratio of AA to (AA + DHA) decreased within 6 h of fumigation from 0.9 to 0.1. Initially, the concentration of (AA + DHA) did not change in the apoplast, but when fumigation was continued, DHA increased and AA remained at a very low constant level. After fumigation was discontinued, DHA decreased very slowly in the apoplast, reaching control level after 70 h. The data show that insufficient AA reached the apoplast from the cytosol to detoxify ozone in the apoplast when the ozone flux into the leaves was 0.9 pmol cm-2 s-1. The transport of DHA back into the cytosol was slower than AA transport into the apoplast. No dehydroascorbate reductase activity could be detected in the apoplast of spinach leaves. In contrast to its extracellular redox state, the intracellular redox state of AA did not change appreciably during a 24-h fumigation period. However, intracellular glutathi-one became slowly oxidized. At the beginning of fumigation, 90% of the total glutathione was reduced. Only 10% was reduced after 24-h exposure of the leaves to 0.3 [mu]L L-1 of ozone. Necrotic leaf damage started to become visible when fumigation was extended beyond a 24-h period. A close correlation between the extent of damage, on the one hand, and the AA content and the ascorbate redox state of whole leaves, on the other, was observed after 48 h of fumigation. Only the youngest leaves that contained high ascorbate concentrations did not exhibit necrotic leaf damage after 48 h.     

Slow pulsatile movements of Schwann cells in vitro: a time-lapse cinemicrographic study

Slow pulsatile movements of Schwann cells in vitro were studied quantitatively by using time-lapse cinemicrography. Schwann cells from peripheral nerves of 3-day-old rats were cultured in serum-free medium. Most Schwann cells showed intermittent episodes of pulsatile movement; each episode consisted of one or several contractile pulses. About half of the episodes consisted of a single pulse, and episodes with more than four pulses were rare. The average episode of activity lasted 2.6 min, while the average duration of a single pulse was 1.5 min. The mean quiescent interval between episodes of activity was 3.7 min. Some cells showed no pulsatile activity. Active cells averaged 6.6 episodes/h. The fraction of time which a Schwann cell spent in pulsatile activity varied widely, with an average of 28%. Behavior of Schwann cells in HEPES-buffered Hanks saline was generally similar to that in the complete medium. Raising K+ to 40 mM or Ca++ to 10 mM did not markedly affect the time course of the pulsatile motility, although the contractions were more vigorous in the high Ca++. Pulsatile movement was reversibly inhibited by cytochalasin B and appeared to be potentiated by drugs that disrupt microtubules.     

Maternal protein homeostasis and milk protein synthesis during feeding and fasting in humans

Little is known about amino acid (AA) and protein metabolism in lactating women. We hypothesized: 1) AA sources other than the plasma acid pool provide substrate for milk protein synthesis in humans and 2) if albumin was one such source, then albumin fractional synthesis rate (FSR) is higher in the lactating women. To test these hypotheses, six healthy exclusively breast-feeding women [27 +/- 3 yr; body mass index (BMI) 26 +/- 2 kg/m2] between 6 wk and 3 mo postpartum and six healthy nonlactating women (28 +/- 2 yr; BMI 22 +/- 1 kg/m2) were studied two times, in random order, during 22 h fasting or 10 h of continuous feeding with a mixed nutrient drink. Protein metabolism was determined using [1-13C]leucine and [15N2]urea. In both the fed and fasted states, a significant portion of milk protein (20 +/- 5 and 31 +/- 6%, respectively) was derived from sources other than the plasma free AA pool. A 70% higher (P < 0.02) FSR of albumin was observed in lactating women during feeding, suggesting that albumin is a likely source of AA for milk protein synthesis. We conclude that plasma free AA contribute only 70-80% of the substrate for milk protein synthesis in humans and that albumin may be a significant source of amino acids for the remainder.     

Arachidonic and docosahexanoic acid content of bovine brain myelin: Implications for the pathogenesis of multiple sclerosis

Lipids were extracted from bovine brain myelin using a mixture of hexane and isopropanol (32). Myelin lipids were resolved, using Sep Pak chromatography, into four fractions: Fraction 1 contained neutral lipids, fraction 2, free fatty acids, fraction 3, ethanolamine phospholipids and fraction 4, choline phospholipids. Docosahexanoic (DHA) and arachidonic (AA) acids in these fractions were measured by RPHPLC. Fraction 2 was analyzed directly, the other three fractions were subjected to alkaline hydrolysis before analysis for DHA and AA. DHA and AA were not found in fraction 1. Both DHA and AA were found in fractions 2 and 3. Only AA was consistently found in fraction 4. These results were confirmed by GC.     

Energie-, Kohlenstoff-, Stickstoff- und Aminosäurenbilanzversuch mit wachsenden Cornish- Hähnen

The time course of AA digestion, AA balance (sV AS), and AA absorption (wV AS) was estimated on growing rats (Wistar rats, LW= 124 g) in different sections of the intestinal tract using the combination of ¹⁵N tracer and TiO₂ marker techniques. The animals received once a diet of ¹⁵N labelled wheat and yeast as protein sources supplemented by TiO₂ as a marker. Up to 6 h after feeding the amino acid composition the ¹⁵N excess and the TiO₂ content in the digesta of stomach, small and large intestine were determinated in the relation of amino acids resp. of ¹⁵N labelled amino acids to the marker. In addition the content of amino acids and the ¹⁵N excess of these amino acids were estimated in plasma. From these data the disappearance rates and the relation of exogenous to endogenous amino acids as well as the sV and the wV values of the different amino acids were calculated for the different gut sections.

The following results were obtained:

- The relative disappearance rate for N and TiO₂ marker out of the stomach went approximately parallel but with a delay for TiO₂ of about 30 minutes.

- The AA composition of the stomach content, the small and the large intestine content did not vary in dependence of the time.

- The AA composition of the stomach digesta was nearly identical to that of the diet, while that of the small intestine was between exogenous AA composition (feed) and endogenous AA composition (digesta on protein free feeding). AA composition of the large intestine digesta showed quite big differences (bacterial AA break down and AA synthesis).

- Considering a delay time (small intestine: 1 h, large intestine: 4 h) the exogenous portion of the different AA remained constant in both of these intestinal sections during the whole experimental time.

- The exogenous AA part varied for small intestine digesta between 31 and 69% (mean value: 41%), and for large intestine digesta between 13 and 39% (mean value: 22%).

- The sV AS values in the small intestine (AA balance resp. precaecal digestibility) differed from 61% (threonine) to 86% (proline) with an average of 73.4 ± 7.4%, those for wV AS (AA absorption) from 81% (lysine) to 94% (proline) with an average of 88.1±4.1%. There were significant differences between AA, but they are negligible for practical purposes.

- In the small intestine the estimated values for postprandial absorption of the exogenous AA accounted after 1 h, 2 h, 4 h and 6 h for 21%, 33.7%, 46.5%, and 70.7% of the AA intake respectively, mean absorption rate = 9.1 ± 0.5%/h.

- The AA balance in the whole tract (sV AS) was 75 to 94% (on average 82 ± 5.3%) and the wV AS (balance corrected by the ¹⁵N method) ranged from 92 to 99% (on average 96±1.8%). These values correspond to the faecal AA digestibility in conventional experiments.

- In the large intestine the postileal disappearance of the AA (sV) was on average 9.7% with a maximum value of 26% for glycine, and with a minimum for methionine of ‐2.1% caused by bacterial synthesis of methionine. Te postileal wV in the large intestine amounted to 7.5%.

- The time course of the disappearance rate of the ¹⁵N labelled AA in the small intestine and the appearance rate of these AA in the plasma showed an analogous behavior. Both of them characterize the postprandial absorption.

The following conclusion can be drawn:

The method used (combination of ¹⁵N tracer and TiO₂ marker techniques) enables determining the time course of transit and the variation of exogenous AA: endogenous AA proportion in the different intestinal sections and estimating the faecal and precaecal digestibility of the different AA.

The course of secretion and absorption of the different AA should be specified in further experiments using the more precise analysis of ¹⁵N by GC‐MS resp. GC‐C‐IRMS technique. An apply of this method to farm animals (pigs) seems to be possible.     

Dynamics of Acetaldehyde Production during Anoxia and Post-Anoxia in Red Bell Pepper Studied by Photoacoustic Techniques

Acetaldehyde (AA), ethanol, and CO2 production in red bell pepper (Capsicum annum L.) fruit has been measured in a continuous flow system as the fruit was switched between 20% O2 and anaerobic conditions. Minimum gas phase concentrations of 0.5 nL L-1, 10 nL L-1, and 1 mL L-1, respectively, can be detected employing a laser-based photoacoustic technique. This technique allows monitoring of low production rates and transient features in real time. At the start of anaerobic treatment respiration decreases by 60% within 0.5 h, whereas AA and ethanol production is delayed by 1 to 3 h. This suggests a direct slow-down of the tricarboxylic acid cycle and a delayed onset of alcoholic fermentation. Reexposure of the fruit to oxygen results in a 2- to 10-fold upsurge in AA production. A short anoxic period leads to a sharp transient peak lasting about 40 min, whereas after numerous and longer anoxic periods, post-anoxic AA production stays high for several hours. High sensitivity of the fruit tissue to oxygen is further evidenced by a sharp decrease in post-anoxic AA production upon an early return to anaerobic conditions. Ethanol oxidation by the "peroxidatic" action of catalase is proposed to account for the immediate post-anoxic AA upsurge.     

Modulation of lymphocyte proliferation by macrophages and macrophages loaded with arachidonic acid

Arachidonic acid (AA) is incorporated and exported by macrophages. This fatty acid is also transferred from macrophages (Mphi) to lymphocytes (LY) in co-culture. This observation led us to investigate the effect of macrophages pre-loaded with AA on concanavalin A (Con A)-stimulated lymphocyte proliferation. The experiments were performed in co-culture. This condition reproduces the in vivo microenvironment in which the modulation of lymphocyte proliferation is dependent on the interaction with macrophages. Lymphocytes obtained from untreated rats or from intraperitoneally thioglycolate-injected rats (THIO-treated) were co-cultured with macrophages from the same rats. Firstly, macrophages were co-cultured for 48 h with Con A-stimulated lymphocytes in different proportions: 0.5, 1, 2.5, 5, 10, 20 and 30% of 5 x 10(5) lymphocytes per well. At 1% proportion, macrophages caused maximum stimulation of lymphocyte proliferation; a four- to five-fold increase, for cells from both thioglycolate-treated and untreated rats, respectively, whereas at 20% it caused maximum inhibition. In addition, 1 or 20% macrophages were pre-loaded with several AA concentrations during a period of 6 h and co-cultured with lymphocytes. At 180 microM AA and 1% macrophages, lymphocyte proliferation was inhibited (by 25%), whereas at 20% macrophages, proliferation was increased, by 25- and three-fold, respectively, for cells from untreated and THIO-treated rats. AA added directly to the medium reduced lymphocyte proliferation, also being toxic to these cells at 100 microM. No toxic effects of AA were observed on macrophages. Additional evidence suggests that nitric oxide production is involved in the modulation of lymphocyte proliferation by AA-pre-loaded macrophages. These findings support the proposition that AA can directly modulate lymphocyte proliferation and the interaction between macrophages and lymphocytes.     

Initial Amino Acid Intake Influences Phosphorus and Calcium Homeostasis in Preterm Infants – It Is Time to Change the Composition of the Early Parenteral Nutrition

Background

Early aggressive parenteral nutrition (PN), consisting of caloric and nitrogen intake soon after birth, is currently proposed for the premature baby. Some electrolyte disturbances, such as hypophosphatemia and hypercalcemia, considered unusual in early life, were recently described while using this PN approach. We hypothesize that, due to its impact on cell metabolism, the initial amino acid (AA) amount may specifically influence the metabolism of phosphorus, and consequently of calcium. We aim to evaluate the influence of AA intake on calcium-phosphorus metabolism, and to create a calculation tool to estimate phosphorus needs.

Methods

Prospective observational study. Phosphate and calcium plasma concentrations and calcium balance were evaluated daily during the first week of life in very preterm infants, and their relationship with nutrition was studied. For this purpose, infants were divided into three groups: high, medium and low AA intake (HAA, MAA, LAA). A calculation formula to assess phosphorus needs was elaborated, with a theoretical model based on AA and calcium intake, and the cumulative deficit of phosphate intake was estimated.

Results

154 infants were included. Hypophosphatemia (12.5%) and hypercalcemia (9.8%) were more frequent in the HAA than in the MAA (4.6% and 4.8%) and in the LAA group (0% and 1.9%); both p<0.001.

Discussion

Calcium-phosphorus homeostasis was influenced by the early AA intake. We propose to consider phosphorus and calcium imbalances as being part of a syndrome, related to incomplete provision of nutrients after the abrupt discontinuation of the placental nutrition at birth (PI-ReFeeding syndrome).We provide a simple tool to calculate the optimal phosphate intake. The early introduction of AA in the PN soon after birth might be completed by an early intake of phosphorus, since AA and phosphorus are (along with potassium) the main determinants of cellular growth.