Free AA Research Articles

Microbiology, Biochemistry, and Volatile Composition of Tulum Cheese Ripened in Goat's Skin or Plastic Bags

Tulum cheeses were manufactured from raw ewe's milk and ripened in goat's skin bags (tulums) or plastic containers to understand the effect of ripening container on the chemical composition, biochemistry, microbiology, and volatile composition of Tulum cheeses during 150 d of ripening. Chemical compositions of the cheeses ripened in tulums were significantly different and the moisture contents decreased rapidly in those cheeses because of the porous structure of the tulum. Higher microbial counts were detected in the cheeses ripened in plastic than in cheeses ripened in tulums. Differences in nitrogenous compounds and total free AA of the cheeses were not significant. Total concentrations of free AA in cheeses increased with age and Glu, Ala, Val, Leu, and Phe were the most abundant AA in the cheeses. Urea-PAGE of pH 4.6-insoluble fractions of the cheeses during ripening showed similar degradation patterns in all cheeses. Peptide profiles by reversed-phase HPLC of pH 4.6- and ethanol-soluble or ethanol-insoluble fractions of the cheeses revealed only minor differences in the concentrations of some peptides among the cheeses; however, age-related changes in peptide concentrations were significantly different among the cheeses. Cheeses were analyzed at 90 d of ripening for volatile compounds by solid-phase microextraction gas chromatography-mass spectrometry. One hundred volatile components were identified, including 11 acids, 16 esters, 12 methyl ketones, 7 aldehydes, 22 alcohols, 7 sulfur compounds, 6 terpenes, and 19 miscellaneous compounds. The main components were short-chain fatty acids, 2-butanone, diacetyl, and primary alcohols. Quantitative differences in several volatile compounds were evident among the cheeses. Cheeses ripened in tulums or plastic had similar aroma patterns, but the concentrations of some components were different.

Comparison of the Compositional, Microbiological, Biochemical, and Volatile Profile Characteristics of Nine Italian Ewes’ Milk Cheeses

Nine Italian ewes’ milk cheeses were compared for compositional, microbiological, biochemical, and volatile profile characteristics. Mean values for the gross composition were rather similar among cheeses. The lowest pH values were found for cheeses that used primary starters. At the end of ripening, cheeses made from raw milk contained >6.0 log10 cfu/g of nonstarter lactic acid bacteria. Several species of lactobacilli were identified, but Lactobacillus plantarum and Lactobacillus paracasei were dominant. Random amplified polymorphic DNA-PCR analysis showed the biodiversity among the strains, and in several cases a relationship with the cheese of provenance. Cheeses differed mainly for secondary proteolysis, as shown by the principal component analysis applied to reversed-phase fast protein liquid chromatography data of the pH 4.6-soluble fractions and by determination of the free AA. A total of 113volatile components were identified in the Italian Pecorino cheeses by solid-phase microextraction coupled with gas chromatography–mass spectrometry analysis. The volatile profiles of the 9 cheeses differed significantly. Quantitatively, alcohols were the most abundant chemical class for some cheeses, whereas ketones were the most abundant for other cheeses. Esters and carboxylic acids were largely found. Specific volatile components seemed to distinguish specific cheeses.

Muscle growth and plasma concentrations of amino acids, insulin-like growth factor-I, and insulin in growing pigs fed reduced-protein diets1,2

Twenty barrows were used to determine if partial replacement of protein-bound AA with crystalline AA (CAA) reduces AA use for muscle tissue and whole-body growth. Barrows (44.2 +/- 1.3 kg of BW) were assigned to 4 diets in a randomized complete block design. Diets consisted of 16.1% CP with no CAA, and 12.8, 10.1, and 7.8% CP containing CAA. As the CP concentration decreased, CAA were gradually increased to meet requirements on a true ileal digestibility basis. Barrows were weighed on d 0 and 13. Blood samples were collected before the morning feeding on d 0, 6, and 12 (prefeeding), and 2 h after the morning feeding on d 13 (postfeeding). Pigs were euthanized on d 13, and liver and right LM were removed and weighed. The reduction in the dietary CP concentration linearly decreased (P < 0.01) ADG, G:F, LM weight, and the CP content of LM. Reducing the CP concentration decreased pre- and postfeeding plasma concentrations of IGF-I (linear, P < 0.01) and insulin (linear, P < 0.10). The reduction in the dietary CP concentration increased prefeeding plasma concentrations of Ala, Gln, Gly, and total AA but decreased Arg, Asn, His, Ile, Phe, Trp, and Tyr (linear, P < 0.05). Plasma concentration of total indispensable AA decreased initially and increased thereafter as the dietary CP concentration decreased from 16.1 to 7.8% (quadratic, P < 0.01). The reduction in the dietary CP concentration increased postfeeding plasma concentrations of Ala, Lys, Met (linear, P < 0.01), and Gly (linear, P = 0.073) and decreased Asn, Ser, Tyr, Arg, His, and Leu (linear, P < 0.05). Plasma concentrations of Ile, Phe, Thr, Trp, and Val decreased initially and increased thereafter as the dietary CP concentration decreased from 16.1 to 7.8% (quadratic, P < 0.05). In muscle tissue, concentrations of free Ala, Asp, Glu, Gln, Gly, and Lys increased (linear, P < 0.05) as the dietary CP concentration decreased. Concentrations of free His, Ile, Phe, Thr, Trp, and Val in muscle tissue decreased initially and increased thereafter as the dietary CP concentration decreased from 16.1 to 7.8% (quadratic, P < 0.05). In summary, the reduction in the dietary protein-bound AA decreased whole-body and LM growth, altered the free AA pool profile in muscle tissue, and decreased plasma insulin and IGF-I. As the replacement of protein-bound AA with CAA increased, 1) free Ala and Gln in muscle tissue increased, indicating an increase of muscle tissue protein breakdown; and 2) utilization of indispensable AA in muscle tissue decreased.

Production of Ingredient-Type Cheddar Cheese with Accelerated Flavor Development by Addition of Enzyme-Modified Cheese Powder

Fast-ripened Cheddar cheeses for ingredient purposes were produced by addition of a dried enzyme-modified cheese (EMC; 0.25 and 1g/100g of milled curd) at the salting stage during a standard Cheddar cheese-making procedure. Populations of starter and nonstarter lactic acid bacteria (NSLAB), levels of proteolysis and lipolysis, volatile analysis, and flavor development (by quantitative descriptive sensory analysis) were monitored over a 6-mo ripening period. Levels of free AA and free fatty acids were elevated in the experimental cheeses on d 1 because of inclusion of the EMC. Counts of NSLAB were also elevated in the experimental cheeses compared with the control cheese from the start of ripening. Levels of free AA were slightly elevated in the experimental cheeses at 1, 2, and 4 mo, but significantly greater accumulations were detected by 6 mo of ripening, with His, Leu, and glutamate reflecting the greatest increases. Levels of long-chain free fatty acids increased up to 2 mo, indicating an initial stimulation of lipolysis, but had decreased by 6 mo, indicating greater catabolism, probably caused by NSLAB and increased starter lysis. Principal component analysis of the volatile compounds showed few differences in the aroma profiles among the cheeses up to 4 mo of ripening, but a large separation of the cheeses supplemented with EMC relative to the control was observed by 6 mo. Sensory analysis of the cheeses with added EMC showed an acceleration of 2 mo in flavor development compared with the control cheese with the addition of 1g/100g of EMC developing a flavor profile at 4 mo similar to the control cheese at 6 mo of ripening. However, atypical Cheddar flavors developed on prolonged storage. This study shows the potential of adding EMC during Cheddar production to produce a fast-ripened ingredient-type Cheddar cheese.

Effect of High-Pressure Treatment and a Bacteriocin-Producing Lactic Culture on the Proteolysis, Texture, and Taste of Hispánico Cheese

The effects of high-pressure treatment, by itself or in combination with a bacteriocin-producing culture added to milk, on the proteolysis, texture, and taste of Hispánico cheese were investigated. Two vats of cheese were manufactured from a mixture of cow and ewe milk. Milk in one vat was inoculated with 0.5% Lactococcus lactis ssp. lactis INIA 415, a nisin Z and lacticin 481 producer; 0.5% L. lactis ssp. lactis INIA 415-2, a bacteriocin-nonproducing mutant; and 2% of a commercial Streptococcus thermophilus culture. Milk in the other vat was inoculated with 1% L. lactis ssp. lactis INIA 415-2 and 2% S. thermophilus culture. After ripening for 15 d at 12°C, half of the cheeses from each vat were treated at 400MPa for 5min at 10°C. Ripening of high-pressure-treated and untreated cheeses continued at 12°C until d 50. High-pressure treatment of cheese made from milk without the bacteriocin producer accelerated casein degradation and increased the free AA content, but it did not significantly influence the taste quality or taste intensity of the cheese. Addition of the bacteriocin producer to milk lowered the ratio of hydrophobic peptides to hydrophilic peptides, increased the free AA content, and enhanced the taste intensity. The combination of milk inoculation with the bacteriocin producer and high-pressure treatment of the cheese resulted in higher levels of both hydrophobic and hydrophilic peptides but had no significant effect on the free AA content, taste quality, or taste intensity.

Altered membrane free unsaturated fatty acid composition in human colorectal cancer tissue

Polyunsaturated free fatty acids (PUFAs) participate in normal functioning of the cell, particularly in control intracellular cell signalling. As nutritional components they compose a human diet with an indirect promoting influence on tumourogenesis. The PUFAs level depends on the functional state of the membrane. This work is focused on changes only of free unsaturated fatty acids amount (AA - arachidonic acid, LA - linoleic acid, ALA - alpha-linolenic acid, palmitoleic acid (PA) and oleic acid) in cell membranes of colorectal cancer of pT3 stage, G2 grade without metastasis. Qualitative and quantitative composition of free unsaturated fatty acids in the membrane was determined by high-performance liquid chromatography. It was shown that the malignant transformation was accompanied by a decrease in amount of LA and ALA while arachidonic and oleic acids increased. It is of interest that free AA levels are elevated in colon cancer, as AA is the precursor to biologically active eicosanoids.

Biochemical and morphological developments are partially impaired in intestinal mucosa from growing pigs fed reduced-protein diets supplemented with crystalline amino acids1

The objective of this study was to determine if a reduction in dietary CP, with partial replacement of the intact protein with crystalline AA (CAA), would alter growth, morphology, and free or peptide-bound AA concentrations of intestinal mucosa in growing pigs. Twenty-four barrows (37.0 +/- 1.5 kg of BW) were fed 1 of 4 diets for 24 d: 16.1% CP with no CAA, or 12.8, 10.1, or 7.8% CP (analyzed values, as-fed) containing CAA. As CP decreased, CAA were gradually increased to meet requirements on a true ileal digestible basis. Pigs were euthanized 2 h postmeal on d 24, and mucosal samples from duodenum, jejunum, and ileum were collected. Reducing dietary CP decreased ADG, G:F, and final weight (linear, P < 0.05). With reduced dietary CP, mucosal protein concentration decreased in the jejunum (quadratic, P < 0.05) and tended to decrease in the ileum (linear, P = 0.062). Reduction of the dietary CP concentration from 16.1 to 7.8% tended to decrease the crypt depth (linear, P < 0.10) and decreased villus width (linear, P < 0.05) in duodenum and jejunum mucosa but did not reduce villus height or villus surface area in any regions of the small intestine. In the duodenum, a reduction in dietary CP increased free Lys, Met, and Thr (linear, P < 0.05) and peptide-bound Lys and Thr (quadratic, P < 0.10). In the jejunum, reducing CP decreased free Cys (linear P < 0.05) and tended to decrease free Asn and His (linear, P < 0.10) and peptide-bound His (quadratic, P = 0.061) and Ile, Leu, and Val (linear, P < 0.10). In the ileum, reducing CP decreased free Asn, Ser, Tyr, Arg, His, Phe (linear, P < 0.05), and Leu (linear, P = 0.054) and peptide-bound Gly and Ser (linear, P < 0.05) and tended to decrease peptide-bound Ile, Leu, Phe, Val (linear, P < 0.10), and Lys (linear P < 0.05). In conclusion, reduced-CP diets supplemented with CAA lead to a reduction in growth performance, associated with biochemical and morphological modifications of the intestinal mucosa.

Indispensable amino acid concentrations decrease in tissues of stomachless fish, common carp in response to free amino acid- or peptide-based diets

The premise that free amino acid or dipeptide based diets will resolve the nutritional inadequacy of formulated feeds for larval and juvenile fish and improve utilization of nitrogen in comparison to protein-based diets was tested in stomachless fish, common carp (Cyprinus carpio L.) larvae. We examined the postprandial whole body free amino acid (FAA) pool in fish that were offered a FAA mixture based diet for the duration of 2 or 4 weeks. We found that the total amount and all indispensable amino acids concentrations in the whole body decreased after a meal. We then fed juvenile carp with dietary amino acids provided in the FAA, dipeptide (PP), or protein (live feed organisms; brine shrimp Artemia salina nauplii, AS) forms. Histidine concentrations in the whole fish body increased in all dietary groups after feeding whereas all other indispensable amino acids decreased in FAA and PP groups in comparison to the AS group. Taurine appears to be the major osmotic pressure balancing free amino acid in larval freshwater fish which may indicate a conditional requirement. We present the first evidence in larval fish that in response to synthetic FAA and PP diets, the whole body indispensable free AA concentrations decreased after feeding. This study shows that amino acids given entirely as FAA or PP cannot sustain stomachless larval fish growth, and may result in depletion of body indispensable AA and most of dispensable AA. The understanding of these responses will determine necessary changes in diet formulations that prevent accelerated excretion of amino acids without protein synthesis.

Characterization of the lipolytic pathways that mediate free fatty acid release during Fas/CD95-induced apoptosis

We have undertaken a study to characterize the lipolytic pathway responsible for the generation of free fatty acids (FFA) during Fas/CD95-induced apoptosis in Jurkat cells. It was initially shown that the cellular lipid fraction that suffered the major quantitative decrease during Fas-induced apoptosis was that of phosphatidylcholine (PC). In addition, the secretion of palmitic acid-derived FFA was largely prevented by D609, an inhibitor of PC-specific phospholipase C (PC-PLC) and also by the diacylglycerol lipase (DAGL) inhibitor RHC-80267, suggesting that the secretion of these FFA during Fas-induced apoptosis is mediated by the generation of DAG by a PC-PLC activity and, sequentially, by a 1-DAGL activity which generates the FFA from its sn-1 position. The endocannabinoid 2-arachidonoyl glycerol (2-AG) should be generated as a sub-product of this pathway, but it did not accumulate inside the cells nor was secreted into the supernatant. Interestingly, the complete inhibition of free AA secretion during Fas-induced apoptosis was only achieved by using the AA trifluoromethylketone, which not only inhibits all types of phospholipase-A(2) (PLA(2)) activities, but also the described lytic activities on 2-AG. Using a combination of RHC-80267 and the iPLA(2)-specific inhibitor bromoenol lactone, it was shown that the DAGL pathway also cooperates with iPLA(2) in the generation of free arachidonate.

Functional characterization of a cloned pig intestinal peptide transporter (pPepT1)

Absorption of dietary protein can be mediated through the uptake of AA as free AA or small peptides. A H(+)-coupled, peptide transport protein, PepT1, is responsible for the absorption of small peptides arising from digestion of dietary proteins in the small intestine. The magnitude of peptide absorption and the nutritional significance of PepT1 are unknown for many food-producing animals; thus, the objective of this study was to clone and determine the functional characteristics of the pig PepT1 (pPepT1). Two cDNA-encoding pPepT1 were isolated, which contain alternative polyadenylation sites. The predicted pPepT1 is a 708-AA protein, which shows 82.8, 85.7, and 64.7% AA identity to human, sheep, and chicken PepT1, respectively. On northern blots, two pPepT1 mRNA of approximately 2.9 and 3.5 kb were detected in the duodenum, jejunum, and ileum of the small intestine and are presumed to result from alternative polyadenylation. Uptake of [(3)H]-Gly-Sar was measured in Chinese hamster ovary cells transiently transfected with a pPepT1 expression vector to study the functional expression of pPepT1. Peptide transport was H(+)-dependent, with an optimal pH of 6.0 to 6.5. The ability of pPepT1 to transport various peptides was assayed by calculating the concentration of unlabeled peptide that inhibited 50% of [(3)H]-Gly-Sar uptake (IC(50)) in transfected cells. Eleven dipeptides and two tripeptides had IC(50) values that ranged from 0.004 to 0.53 mM. Three peptides, Lys-Lys, Arg-Lys, and Lys-Trp-Lys, had IC(50) values greater than 1. 38 mM and seem to be poor substrates for pPepT1. For all three tetrapeptides examined, uptake of Gly-Sar was too small to measure, even at a concentration of 10 mM tetrapeptide; therefore, IC(50) values could not be calculated. These results demonstrate that pPepT1 can transport a variety of dipeptides and tripeptides but not tetrapeptides.

Effects of wilting and mechanical conditioning on proteolysis in sainfoin (Onobrychis viciifolia Scop) wilted herbage and silage

AbstractEnsiling legumes allows conservation of high‐quality forage. However, severe protein degradation occurs during ensiling of legumes. A field study was carried out in 1999 on sainfoin (Onobrychis viciifolia Scop) to investigate the effects of wilting and mechanical conditioning on proteolysis in wilted herbage and silage. The herbage was cut with both a rubber roll conditioning machine (C) and with a mower without conditioner (U). It was wilted in the field to three dry matter (DM) levels (220, 330 and 440 g kg−1) and ensiled in laboratory glass silos. The herbages and the silages were analysed for ensilability characteristics, fermentation quality and nitrogen fractions. During wilting the non‐protein nitrogen (NPN) values increased (p &lt; 0.05) in both U and C treatment. The free amino acid (free AA) values increased (p &lt; 0.05) from 2.4 to 12.9 and from 2.4 to 7.2 mol kg−1 total nitrogen, in the U and C treatments, respectively. Low wilted silages (DM content &lt;320 g kg−1) underwent butyric acid fermentation and extensive proteolysis in both treatments, with lower (p &lt; 0.05) NPN and free AA values in the C treatment. Mechanical conditioning may limit protein breakdown and catabolism of some essential amino acids by reducing the field wilting time in both sainfoin wilted herbage and silage. Copyright © 2004 Society of Chemical Industry

Plant capture of free amino acids is maximized under high soil amino acid concentrations

Free amino acids (AA's) represent a significant source of available N for some plants and soil microorganisms. It can be expected, however, that significant competition will exist between plants and microorganisms for this organic N resource. Our study indicated that microbial capture and utilization of glycine was very rapid at a range of soil solution concentrations (0.1 μM to 10 mM) indicating that significant competition will exist between roots and soil microorganisms. Plant capture of free AA's was maximal at high soil solution concentrations where microbial utilization was slowest. Our results suggest that plant capture of soil dissolved organic N may primarily occur in organic rich patches in soil where concentrations of free AA's are high.

Ruminal and host adaptations to changes in frequency of protein supplementation.

The effect of altering supplementation frequency on host N balance and key N transactions in the ruminal ecosystem were monitored. Four ruminally fistulated beef steers (BW = 513 kg; SEM = 6.5) were used in a 2 x 2 crossover design with two periods and two supplementation frequency treatments. Supplementation frequencies were 2 and 7 d/wk. Steers were fed tallgrass prairie hay (73.1% NDF, 5.3% CP) ad libitum. Supplement (42% CP; DM basis) was fed at 0.36% BW/d to steers supplemented 7 d/wk. Steers supplemented 2 d/wk received the same amount of supplement per week, but it was equally split among the two supplementation events. Steers supplemented 7 d/wk had higher forage (P < 0.02) and total digestible OM intake (P < 0.06), total N intake, fecal N excretion, and N retention. Although both supplementation frequencies were characterized by positive N balance, the decrease in N retention in the steers supplemented 2 d/wk was due to higher (P < 0.01) urinary N loss. Ruminal fluid was sampled at 0, 2, 4, 6, 12, 24, 48, and 72 h after supplementation beginning on a day when both treatments were supplemented. Frequency x hour interactions (P < 0.02) were observed for ruminal N metabolism criteria. Counts of peptide- and AA-fermenting bacteria peaked at 2 h and returned to nadir by 12 h for steers supplemented 7 d/wk. Steers supplemented 2 d/wk peaked at 6 h with a greater population and returned to nadir at 48 h. Ruminal ammonia concentrations followed a similar trend. Specific activity of ammonia production was lower (P < or = 0.05) immediately after supplementation for steers supplemented 2 d/wk, but by 12 h was the same as for 7 d/wk steers. Ruminal peptides and free AA peaked at 2 h for steers supplemented 2 d/wk and were generally higher (P < or = 0.05) during the first 6 h compared with steers supplemented 7 d/wk. Total VFA concentration was not different (P = 0.35) due to supplementation frequency. Frequency x hour interactions (P < 0.01) were observed for all molar proportions of VFA. The molar proportion of acetate and acetate:propionate ratio were lower (P < 0.01) and the molar proportions of propionate and butyrate were higher for steers supplemented 2 d/wk from 4 h to 24 h. In conclusion, forage use and N balance improved with supplementation 7 d/wk, but supplementation 2 d/wk was associated with some desirable shifts in select ruminal events that may contribute to moderating potential negative impacts of supplementing infrequently.

Apparent ruminal degradation and rumen escape of soluble nitrogen fractions in grass and grass silage administered intraruminally to lactating dairy cows1

The main objective of this study was to investigate in vivo ruminal degradation and rumen escape of soluble N fractions in grass and grass silage. Soluble protein and long-chain peptides (PLP), small peptides (SP) and free AA (FAA) were obtained from fresh grass and grass silages fertilized with different levels of N. Soluble extracts from the forages were pulse dosed into the rumen of three cannulated lactating dairy cows, and a simple or complex model was used to examine the kinetics of the soluble N fractions in the rumen. When soluble extracts from silage were investigated, pulse dosages of total nonammonia N (NAN) were 21, 27, and 32 g, while for fresh grass only dosages of 20 g were ruminally administered. In the silage extracts, mean proportions of PLP-N, SP-N, and FAA-N in the NAN were 30, 52, and 18%, respectively, whereas in the fresh grass the corresponding values were 67, 20, and 13%. From silage extracts, all three soluble N fractions showed a linear decrease (P < 0.05) in degradation rate and an increase (P < 0.05) in ruminal escape with increasing dosage. In silage, mean degradation rates, parameterized from the complex model, were 230, 214, and 334%/h for PLP-N, SP-N, and FAA-N, respectively, and the ruminal escape was highest (P < 0.05) for SP-N (11.2% of dose) and lowest (P < 0.05) for FAA-N (5.0% of dose). No differences in degradation rate and ruminal escape between fresh grass and silage were observed. However, the proportion of N dose converted to ammonia was only 24% in the fresh grass, whereas for the silages a mean value of 76% was found. From this study, it is concluded that a significant amount of dietary soluble N escapes ruminal degradation, and thus contributes to the intestinal AA supply. Moreover, if the main aim is to study degradation kinetics of individual N fractions, a complex model should be used in the evaluation. This model can also be used to study ruminal synchronization of N and energy for microbial growth.

Apparent ruminal degradation and rumen escape of soluble nitrogen fractions in grass and grass silage administered intraruminally to lactating dairy cows.

The main objective of this study was to investigate in vivo ruminal degradation and rumen escape of soluble N fractions in grass and grass silage. Soluble protein and long-chain peptides (PLP), small peptides (SP) and free AA (FAA) were obtained from fresh grass and grass silages fertilized with different levels of N. Soluble extracts from the forages were pulse dosed into the rumen of three cannulated lactating dairy cows, and a simple or complex model was used to examine the kinetics of the soluble N fractions in the rumen. When soluble extracts from silage were investigated, pulse dosages of total nonammonia N (NAN) were 21, 27, and 32 g, while for fresh grass only dosages of 20 g were ruminally administered. In the silage extracts, mean proportions of PLP-N, SP-N, and FAA-N in the NAN were 30, 52, and 18%, respectively, whereas in the fresh grass the corresponding values were 67, 20, and 13%. From silage extracts, all three soluble N fractions showed a linear decrease (P < 0.05) in degradation rate and an increase (P < 0.05) in ruminal escape with increasing dosage. In silage, mean degradation rates, parameterized from the complex model, were 230, 214, and 334%/h for PLP-N, SP-N, and FAA-N, respectively, and the ruminal escape was highest (P < 0.05) for SP-N (11.2% of dose) and lowest (P < 0.05) for FAA-N (5.0% of dose). No differences in degradation rate and ruminal escape between fresh grass and silage were observed. However, the proportion of N dose converted to ammonia was only 24% in the fresh grass, whereas for the silages a mean value of 76% was found. From this study, it is concluded that a significant amount of dietary soluble N escapes ruminal degradation, and thus contributes to the intestinal AA supply. Moreover, if the main aim is to study degradation kinetics of individual N fractions, a complex model should be used in the evaluation. This model can also be used to study ruminal synchronization of N and energy for microbial growth.

Cortical impact injury in rats promotes a rapid and sustained increase in polyunsaturated free fatty acids and diacylglycerols.

Neurotrauma activates the release of membrane phospholipid-derived second messengers, such as free arachidonic acid (20:4n-6, AA) and diacylglycerols (DAGs). In the present study, we analyze the effect of cortical impact injury of low-grade severity applied to the rat frontal right sensory-motor cortex (FRC) on the accumulation of free fatty acids (FFAs) and DAGs in eight brain areas 30 min and 24 hours after the insult. At these times, accumulation of FFAs and DAGs occurred mainly in the damaged FRC. The cerebellum was the only other brain area that displayed a significant accumulation of DAGs by day one post-injury. By 30 min, accumulation of free AA in the FRC displayed the greatest relative increase (300% over sham value), followed by free docosahexaenoic acid (22:6n-3, DHA, 150%), while both 20:4-DAGs and 22:6-DAGs were increased 100% over sham values. At day one, free 22:6 and 22:6-DAGs showed the greatest increase (590% and 230%, respectively). These results suggest that TBI elicits the hydrolysis of phospholipids enriched in excitable membranes, targeting early on 20:4-phospholipids (by 30 min post- trauma) and followed 24 hours later by preferential hydrolysis of DHA-phospholipids. These lipid metabolic changes may contribute to the initiation and maturation of neuronal and fiber track degeneration observed following cortical impact injury.

IL-13 induces serine phosphorylation of cPLA2 in mouse peritoneal macrophages leading to arachidonic acid and PGE2 production and blocks the zymosan-induced serine phosphorylation of cPLA2 and eicosanoid production

In a recent investigation, we demonstrated that long-term treatment of macrophages with IL-13 enhances cPLA2 expression and modulates zymosan-stimulated AA mobilization. In the present study, we examine the ability of IL-13 to modify the cPLA2 activity and the AA mobilization of macrophages after a short-period of treatment. We demonstrate that in resting macrophages, IL-13 induces, through a MAP kinase-dependent process, (1) an increase of free AA release within 15 min, followed by increased PGE2 production and (2) a time-dependent serine phosphorylation of cPLA2. Conversely, in macrophages stimulated by zymosan, IL-13 added 30 min before zymosan inhibited the AA release and the serine phosphorylation of cPLA2 induced by the phagocytic agonist. In conclusion, these findings show for the first time that a Th2-type cytokine can upregulate cPLA2 activity and downregulate zymosan-induced AA metabolism. Thus, establishment of the connection between these two events may help to understand the complex regulatory role of IL-13 on the macrophage AA metabolism.

Cell-cell interaction between platelets and IL-1β-stimulated vascular smooth muscle cells in synthesis of thromboxane A 2

Transcellular biosynthesis of thromboxane (Tx) A 2 between vascular smooth muscle cells (SMC) and platelets has been investigated by using 14C-arachidonic acid (AA) radiolabeled rat SMC (or platelets) and the fate of the label in phospholipids and eicosanoid fractions was studied using radioimmunoassay (RIA) and thin-layer chromatography (TLC). Stimulation of SMC with interleukin-1β (IL-1β) resulted in production of cyclooxygenase metabolites (e.g. 6-keto-PGF 1α, PGE 2, PGF 2α, PGD 2), 15-, 11-, 5-HETE, and free AA, with a coincident decline of phosphatidylcholine (PC) in SMC. IL-1β did not induce TXB 2 production, a stable metabolite of TXA 2 measured by TLC and radioimmunoassay, either in human platelets from 0.01–100 U/ml for 1 h or in SMC for 24 h. However, human platelets converted exogenous PGH 2 to TXA 2 despite cyclooxygenase inhibition or PGH 2 receptor blockade. Furthermore, TXB 2 was produced in large quantities during co-incubation of IL-1β-stimulated SMC with human platelets for 30 min in concert with a significant decrease of 6-keto-PGF 1α and eicosanoids (PGE 2, PGF 2α and PGD 2) compared with control ( P < 0.01). Pretreatment of SMC with cycloheximide and actinomycin not only inhibited IL-1β-induced eicosanoid synthesis and phospholipid breakdown but also diminished TXB 2 production when co-incubated with platelets. These data suggest that a cell-cell interaction, i.e. platelet utilizing SMC-derived endoperoxides for its TXA 2 production, might cause an excess thromboxane A 2 synthesis.

Degradation of Protein and Utilization of the Hydrolytic Products by a Predominant Ruminal Bacterium, Prevotella ruminicola B14

Degradation and utilization of protein by Prevotella ruminicola B14, a proteolytic bacterium that is prominent in the rumen, was examined. In preliminary experiments, proteinaceous N sources produced faster growth rates than did NH4Cl, based on changes in optical density over time. However, ammonium chloride produced a greater maximum cell density than did proteinaceous N sources. Of the proteinaceous N sources, an enzymatic hydrolysate of soybean protein with a relative peptide size of 3 AA residues produced a greater growth rate and maximum cell density compared with the other proteinaceous N sources. Further experiments revealed that P. ruminicola B14 grew faster and to a greater final dry weight with soybean protein than with casein. Degradation of both proteins was low as was indicated by the slow disappearance of soluble protein, low concentrations of free AA and peptides, and the decrease in ammonia concentrations over time. Patterns of degradation did differ between the two proteins, however. Accumulation of peptides and free AA from soybean protein peaked 2h earlier than those from casein, and concentrations of free AA and peptides from soybean protein were lower on average than those from casein. Prevotella ruminicola B14 preferentially utilized Asp, Ile, Leu, Lys, and Arg from soybean protein compared with casein. The relative size of peptides that accumulated from both proteins, as determined by the ratio of ninhydrin reaction after HCl hydrolysis to ninhydrin reaction before HCl hydrolysis, suggested that part of the proteolytic activity of P. ruminicola B14 is a dipeptidase. Our findings suggest that P. ruminicola may have a greater impact on peptide degradation than on protein degradation in the rumen.

Effect of intravenous histidine or histidine peptide infusion on milk protein yield in lactating goats with an induced histidine deficiency

Studies involving infusion of stable isotope labelled peptides have shown that the mammary gland has the ability to utilise peptide-derived AA for milk protein synthesis (Backwell et al., 1994a) and that peptides may be involved in the supply of phenylalanine to the mammary gland in vivo (Backwell et al., 1994b). The aim of the present experiment was to compare milk production responses to systemic (jugular vein) provision of histidine as free AA or as a peptide (glycyl-histidine) in lactating dairy goats with an induced histidine deficiency.