Quantitative determination of aromatic amino acids and related compounds in rumen fluid by high-performance liquid chromatography

A rapid method for the quantitative determination of tyrosine (Tyr), phenylalanine (Phe), p-hydroxybenzoic acid (HBA), p-hydroxyphenylacetic acid (HPA), benzoic acid (BZA), p-hydroxyphenylpyruvic acid (HPY), phenylacetic acid (PAA), phenyllactic acid (PLA), tryptophan (Trp), indoleacetic acid (IAA), phenylpyruvic acid (PPY), phenylpropionic acid (PPA) and cinnamic acid (CNA) in goat rumen fluid was established by high-performance liquid chromatography (HPLC). The mobile phase used for isocratic elution was 50 mM sodium phosphate buffer (pH 6.5)–methanol (97:3, v/v). The flow-rate was 1.0 ml/min; column temperature 40°C and compounds were monitored at 215 nm with a UV absorbance detector after injection of 10 μl of filtered rumen fluid. Analysis was completed within 40 min. The minimum detectable limits of quantification (μM) of these compounds were Tyr, 2; Phe, 3; HBA, 1; HPA, 2; BZA, 2; HPY, 8; PAA, 3; PLA, 4; Trp, 2; IAA, 2; PPY, 15; PPA, 8 and CNA, 4. Detectable levels of Tyr, Phe, HPA, BZA, HPY, PAA, PLA, Trp and PPA were found in the deproteinized rumen fluid of goat fed a haycube and concentrate mixture. PAA was the predominant compound before and after feeding. The concentrations of HPA, BZA, PAA, PLA and PPA in the goat rumen fluid increased after feeding, while the concentration of Tyr decreased. Phe, HPY and Trp were minor components at all times. PPY, IAA and CNA were not detected and HBA was not completely resolved in the goat rumen fluid.     

Synthesis of phenylalanine and production of other related compounds from phenylpyruvic acid and phenylacetic acid by ruminal bacteria,protozoa, and their mixture in vitro

Phenylalanine (Phe) synthesis and the production of other related compounds by mixed ruminal bacteria (B), protozoa (P), and a combination of the two mixture (BP) in an in vitro system were quantitatively investigated using phenylpyruvic acid (PPY) and phenylacetic acid (PAA) as substrates. Rumen microorganisms were collected from fistulated goats fed lucerne cubes (Medicago sativa) and a concentrated mixture twice a day. Microbial suspensions were anaerobically incubated at 39 degrees C for 12 h. Phe and some other related compounds in both supernatants and microbial hydrolysates of the incubations were analysed by HPLC. A large quantity of Phe was produced from both PPY and PAA not only in B but also in P. In B suspensions, free Phe also accumulated in the medium only when PPY was used as a substrate. The ability of B to synthesize Phe from both PPY and PAA (expressed as unit 'per microbial nitrogen') was 5.1 and 24.8% higher than P, respectively. Phe production from PPY in B and P was 43.5 and 55.2% higher than that from PAA. Large amounts of PAA (17-27%) were produced from PPY in all microbial suspension and production amounts were similar in B and P. Small amounts of benzoic acid (BZA) were produced from PPY and PAA in B, P, and BP, and higher BZA production was observed in P as compared to B. Phenylpropionic acid (PPR) was produced in B from both PPY and PAA, but not in P or BP. A trace amount of phenyllactic acid (PLA) was detected only from PPY in B. Higher concentrations of an unknown compound from PPY and PAA were found to be accumulated in the body protein of B and also in the medium of P, and production of the compound from both PPY and PAA was also higher in B than P.     

In vitro metabolism of phenylalanine by ruminal bacteria,protozoa, and their mixture

An in vitro study was conducted to examine the metabolism of phenylalanine (Phe) by mixed rumen bacteria (B), mixed rumen protozoa (P), and a combination of the two (BP). Rumen microorganisms were collected from fistulated goats fed lucerne cubes (Medicago sativa) and a concentrated mixture twice a day. Microbial suspensions were anaerobically incubated at 39 degrees C for 12 h. Phe and some other related compounds in both supernatants and microbial hydrolysates of the incubations were analysed by HPLC. The net degradation rate (&mgr;mol/g microbial nitrogen) of Phe in B was about 1.5-fold higher than that in P. Phe was converted mainly into phenylacetic acid (PAA) and unknown compound(s) that presumably involved tyrosine in B, P, and BP during the 12 h incubation period. Small amounts of benzoic acid (BZA), and traces of phenylpropionic acid (PPR) and phenyllactic acid (PLA) were also produced from Phe. PAA production in B was found to be higher than that in P, whereas it was significantly higher in BP. Although BZA production was less than one-tenth that of PAA production, it was higher in P than in B and BP. PPR was detected in both B and BP, but not in P. PLA was detected only in B. The production of unknown compound(s) was higher in B than in P and BP.     

Effects of salinomycin and vitamin B(6) on in vitro metabolism of phenylalanine and its related compounds by ruminal bacteria,protozoa and their mixture

An in vitro study was conducted to examine the effects of salinomycin (SL) and vitamin B(6) (B(6)) on the production of phenylalanine (Phe) from phenylpyruvic acid (PPY) and phenylacetic acid (PAA) and of PAA from Phe and PPY by mixed rumen bacteria (B), mixed rumen protozoa (P) and their mixture (BP). Rumen microorganisms were collected from fistulated goats fed lucerne cubes (Medicago sativa) and a concentrate mixture (3 : 1) twice a day. Microbial suspensions were anaerobically incubated at 39 degrees C for 12 h. Phe and some other related compounds in both supernatants and microbial hydrolysates of the incubations were analyzed by HPLC. When PPY was used as a substrate, it completely disappeared without additives and converted mainly to Phe and PAA on the average by 396 and 178, 440 and 189, and 439 and 147 &mgr;M in B, P and BP, respectively, during the 12 h incubation period. The rate of disappearance showed no significant differences between the microbial suspensions with and without SL and B(6) during the incubation period. The production of Phe from PPY with SL was enhanced (p<0.05) by 40, 20 and 19% in B, P and BP, respectively, while PAA production from PPY with SL was inhibited (p<0.05) by 35, 37 and 38% in B, P and BP, respectively, during the 12 h incubation period. On the other hand, with B(6), the production of Phe and PAA from PPY tended to be enhanced by 14 and 17, 9 and 11, and 7 and 22% in B, P and BP, respectively, during the 12 h incubation period. When PAA added as a substrate was incubated in the incubation medium without any additives, it disappeared by 483, 462 and 507 &mgr;M and converted mainly to Phe on the average by 231, 244 and 248 &mgr;M in B, P and BP, respectively. The disappearance of PAA with SL was inhibited (p<0.05) by 16, 15 and 20%, in B, P and BP, respectively, whereas the disappearance of PAA with B6 was almost the same as that without B(6) in B and BP suspensions but tended to be enhanced by more than 9% in P suspensions during the 12 h incubation period. The production of Phe from PAA with SL tended to be inhibited by 12, 11 and 8% in B, P and BP, respectively, during the 6 h incubation period, but the inhibition was weakened during the 12 h incubation period, whereas Phe production from PAA with B(6) tended to be enhanced by 13, 16 and 8% in B, P and BP, respectively. When Phe was added as a substrate, the net Phe disappearance without additives was 549, 365 and 842 &mgr;M and converted mainly to PAA on the average by 254, 205 and 461 &mgr;M in B, P and BP, respectively. The net disappearance of Phe with SL was inhibited (p<0.05) by 38, 28 and 46%, whereas the net disappearance of Phe with B(6) was enhanced (p<0.05) by 9, 8 and 7% in B, P and BP, respectively. The production of PAA from Phe with SL was inhibited (p<0.05) by 73, 54 and 76% in B, P and BP, respectively. On the other hand, with B(6), PAA production from Phe was enhanced (p<0.05) by 19, 18 and 20% in B, P and BP, respectively. Based on these results, it seems that SL inhibited Phe disappearance and enhanced the synthesis of Phe from PPY, though not from PAA, and accumulated free Phe in the medium, whereas B(6) also enhanced Phe synthesis both from PPY and PAA, which could provide additional amino N for animals.     

Convenient method for the determination of arginine and its related compounds in rumen fluid by reversed-phase high-performance liquid chromatography

In order to clarify arginine (Arg) metabolism by rumen microorganisms and by the tissues of ruminant animals, a convenient method for the simultaneous determination of Arg, citrulline (Cit), ornithine (Orn), proline (Pro) and 5-aminovaleric acid (5AV), and 4-aminobutyric acid (4AB) and lysine (Lys), incidentally, in goat rumen fluid was established by reversed-phase high-performance liquid chromatography (RP-HPLC). The separation was carried out by stepwise isocratic elution with two mobile phases (solvent A and solvent B) on a LiChrospher 100 RP-18 column (150×4.6 mm I.D., 5 μm particle size) equipped with a guard column (4.0×4 mm, 5 μm particle size). Solvent A is composed of acetonitrile–sodium citrate buffer (pH 7.2) (15:85, v/v) containing tetrahydrofuran (5 ml/100 ml), with solvent B comprising acetonitrile–sodium citrate buffer (pH 5.4) (40:60, v/v). Five compounds (Cit, Arg, Pro, 4AB and 5AV) were separated within 33 min in solvent A and the other two (Orn and Lys) in solvent B. Solvent A was automatically switched to solvent B with the help of a valve controller. Complete separation needs 62 min after sample injection in a single chromatogram. Samples were derivatized with 9-fluorenylmethyloxycarbonyl chloride (FMOC-Cl) and detected on a fluorescence detector at excitation and emission wavelengths of 263 and 611 nm, respectively. The minimum detectable concentrations (μM) (signal-to-noise ratio, S/N 3:1) of these compounds were: 0.65 for Cit, 0.65 for Arg, 1.9 for Pro, 1.3 for 4AB, 1.9 for 5AV, 0.12 for Orn and 0.48 for Lys. When applied to rumen fluid from goats, recoveries of all compounds added to the rumen fluid were 96.6–100.6% for an intra-day study and 93.9–99.4% for inter-day (5 days) studies. The average contents of Orn, 5AV and Lys in the rumen fluid of three goats before morning feeding were 7.3, 13.5 and 3.6 μM, but Cit, Arg, Pro, and 4AB were not found, although all these four compounds were detected 1 h after feeding. Pro (390 μM) and 5AV (497.6 μM) were highest 1 h after feeding and then decreased. Orn levels before morning feeding were most similar to those after feeding.     

Production of tyrosine and other aromatic compounds from phenylalanine by rumen microorganisms

Summary Rumen contents from three fistulated Japanese native goats fed Lucerne hay cubes (Medicago sativa) and concentrate mixture were collected to prepare the suspensions of mixed rumen bacteria (B), mixed protozoa (P) and a combination of the two (BP). Microbial suspensions were anaerobically incubated at 39°C for 12h with or without 1 MM ofl-phenylalanine (Phe). Phe, tyrosine (Tyr) and other related compounds in both supernatant and microbial hydrolysates of the incubations were analyzed by HPLC. Tyr can be produced from Phe not only by rumen bacteria but also by rumen protozoa. The production of Tyr during 12h incubation in B (183.6 mol/g MN) was 4.3 times higher than that in P. One of the intermediate products between Phe and Tyr seems to bep-hydroxyphenylacetic acid. The rate of the net degradation of Phe incubation in B (76.O mol/g MN/h) was 2.4 times higher than in P. In the case of all rumen microorganisms, degraded Phe was mainly (>53%) converted into phenylacetic acid. The production of benzoic acid was higher in P than in B suspensions. Small amount of phenylpyruvic acid was produced from Phe by both rumen bacteria and protozoa, but phenylpropionic acid and phenyllactic acid were produced only by rumen bacteria.     

Phenylalanine and its metabolites induce embryopathies in mouse embryos in culture

The aim of this study was to determine the teratogenicity of phenylalanine (Phe) and Phe metabolites in neurulating mouse embryos. Therefore, the system of whole embryo culture was employed and D9 (neurulating) mouse embryos were exposed to Phe, phenylethylamine (PEA), phenylpyruvic acid (PPA), phenylacetic acid (PAA), 2-OH phenylacetic acid (2-OH PAA), and phenyl-lactic acid (PLA) at concentrations ranging from 0.01 mM to 10 mM for 24 hours. After 24 hours, embryos were examined for morphological abnormalities and protein content by the Lowry method. Phe at 1 and 6 mM concentrations was not teratogenic; however, 10 mM inhibited cranial neural tube closure in 82% of the embryos. PEA was the most toxic factor and concentrations of 1 and 10 mM were embryo-lethal, whereas neural tube closure defects (NTDs) were observed in 67% of the embryos at 0.1 mM. 2-OH PAA was the second most toxic metabolite with concentrations of 1 and 10 mM producing NTDs in 10 and 100% of the embryos, respectively. PLA and PAA produced no NTDs at concentrations of 1 mM, 60% at 5 mM, and 100% at 10 mM. Finally, PPA produced approximately 50% NTDs at both 1 mM and 10 mM concentrations. PLA, PAA, 2-OH PAA, and PPA produced a significant reduction in embryonic protein, and PEA and 2-OH PAA reduced yolk sac protein values. PEA, 2-OH PAA, PPA, PAA, and PLA also produced craniofacial abnormalities, i.e., incomplete expansion of the forebrain, collapse of the optic vesicle, and hypoplasia of the mandible and/or the maxilla.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of biosynthetic intermediates and citrate on the phenyllactic and hydroxyphenyllactic acids production by Lactobacillus plantarum CRL 778

Aim: To evaluate the influence of biosynthetic precursors, intermediates and electron acceptors on the production of antifungal compounds [phenyllactic acid (PLA) and hydroxyphenyllactic acid (OH‐PLA)] by Lactobacillus plantarum CRL 778, a strain isolated from home‐made sourdough. Methods and Results: Growth of fermentative activity and antifungal compounds production by Lact. plantarum CRL 778 were evaluated in a chemically defined medium (CDM) supplemented with biosynthetic precursors [phenylalanine (Phe), tyrosine (Tyr)], intermediates [glutamate (Glu), alpha‐ketoglutarate (α‐KG)] and electron acceptors [citrate (Cit)]. Results showed that the highest PLA production (0·26 mmol l^?1), the main antifungal compound produced by Lact. plantarum CRL 778, occurred when greater concentrations of Phe than Tyr were present. Both PLA and OH‐PLA yields were increased 2‐folds when Cit was combined with α‐KG instead of Glu at similar Tyr/Phe molar ratio. Similarly, glutamate dehydrogenase (GDH) activity was significantly (P < 0·01) stimulated by α‐KG and Cit in Glu‐free medium. Conclusion: Phe was the major stimulant for PLA formation; however, Cit could increase both PLA and OH‐PLA synthesis by Lact. plantarum CRL 778 probably due to an increase in oxidized NAD⁺. This effect, as well as the GDH activity, was enhanced by α‐KG and down regulated by Glu. Significance and Impact of the Study: This is the first study where the role of Glu and GDH activity in the PLA and OH‐PLA synthesis was evidenced in sourdough lactic acid bacteria (LAB) using a CDM. These results contribute to the knowledge on the antifungal compounds production by sourdough LAB with potential applications on the baked goods.     

Simple method for the simultaneous analysis of pipecolic acid and lysine by high-performance liquid chromatography and its application to rumen liquor and plasma of ruminants

A high-performance liquid chromatography method for the simultaneous determination of pipecolic acid (Pip) and lysine (Lys), a precursor of Pip, in the rumen liquor and plasma of ruminant animals was established. Samples of rumen liquor and plasma were deproteinized with 50% acetonitrile and derivatized with a fluorescent agent 9-fluorenylmethyloxy carbonyl chloride (Fmoc-Cl). Chromatographic separation was achieved on a TSK gel ODS-80TM column using a reversed-phase gradient elution system. For the gradient elution, two mobile phases, A and B, were needed, both commonly consisted of: 5 mM l-proline, 2.5 mM cupric sulfate and 6.5 mM ammonium acetate. Mobile phase B additionally contains 50% (v/v) acetonitrile. The pH of both mobile phases was adjusted to 7.0. Derivatized Pip and Lys were detected on a fluorescent detector at excitation and emission wavelengths of 260 and 313 nm, respectively. The calibration curves were linear within the range 0 to 1 mM (r>0.999). The average recoveries for Pip and Lys were 95.9±1.8 and 93.2±2.5% in rumen liquor and 98.3±1.4 and 97.5±1.3% in plasma, respectively. The limits of detection for Pip and Lys were 0.6 and 0.7 μM in rumen liquor and 0.01 and 0.05 μM in plasma. The assay has acceptable precision, relative standard deviation (RSD) for reproducibility (within-day and day-to-day variation) were less than 5.2% for aqueous (5.0 μM Pip and Lys), MB9 (5.0 μM Pip and Lys), plasma (7.1 μM Pip and 85.6 μM Lys) and rumen liquor (28.4 μM Pip and 10.2 μM Lys) samples. The levels of Pip and Lys in faunated goats, determined from three animals over a period of two days sampling, were found to be 36.8±18.1 and 14.6±2.8 μM in rumen liquor, and 7.3±2.5 and 137.3±38.0 μM in plasma at 1 h after feeding. This is the first report on the normal levels of Pip in the rumen liquor and plasma of faunated goat.     

Convenient method of threonine, methionine and their related amino compounds by high-performance liquid chromatography and its application to rumen fluid

A high-performance liquid chromatographic procedure for the quantitative determination of cysteine (Cys), homocysteine (Hcys), methionine sulfoxide (MSO), methionine sulfone (MSO₂), homoserine (Hser), glycine (Gly), threonine (Thr), 2-aminobutyric acid (2AB), methionine (Met), cystathionine (Cysta) and its application to rumen fluid are described. The samples containing Thr, Met and other related amino compounds were derivatized with 9-fluorenylmethyl chloroformate. The separation of compounds was accomplished with a methanol gradient in 25 mM sodium citrate buffer (obtaining pH 6.40 and 3.80 by addition of 25 mM citric acid). All derivatized compounds were separated on a Mightysil RP-18 GP (150×4.6 mm I.D., 5 μm particle size) column. All analytes were detected at 265 nm with UV detection. The limits of detection (μM) (S/N ratio, 3:1) and quantification (μM) (S/N ratio, 10:1) of Cys, Hcys, MSO, MSO₂, Hser, Gly, Thr, 2AB, Met and Cysta were 0.50 and 1.68; 1.76 and 5.85; 0.85 and 2.88; 0.92 and 3.09; 1.04 and 3.52; 0.76 and 2.52; 0.65 and 2.18; 0.39 and 1.36; 0.31 and 1.03; 0.17 and 0.58, respectively. The recoveries of all compounds in rumen fluid were 97.93–102.3% in the within-day study and 94.52–98.69% on different day (6 days) studies. The average contents (μM) of Cys, Gly, Thr, 2AB, Met and Cysta were 1.72, 45.6, 20.0, 4.3, 2.11 and 3.42 before morning feeding. The concentration of Thr, 2AB and Cysta in rumen fluid tended to increase with time after feeding whereas Met showed the opposite tendency.     

Phospholipase A2 inhibitors synthesized by two entomopathogenic bacteria, Xenorhabdus nematophila and Photorhabdus temperata subsp. temperata

The entomopathogenic bacteria Xenorhabdus nematophila and Photorhabdus temperata subsp. temperata suppress insect immune responses by inhibiting the catalytic activity of phospholipase A(2) (PLA(2)), which results in preventing biosynthesis of immune-mediating eicosanoids. This study identified PLA(2) inhibitors derived from culture broths of these two bacteria. Both X. nematophila and P. temperata subsp. temperata culture broths possessed significant PLA(2)-inhibitory activities. Fractionation of these bacterial metabolites in the culture broths using organic solvent and subsequent chromatography purified seven potent PLA(2) inhibitors, three of which (benzylideneacetone [BZA], proline-tyrosine [PY], and acetylated phenylalanine-glycine-valine [FGV]) were reported in a previous study. Four other compounds (indole, oxindole, cis-cyclo-PY, and p-hydroxyphenyl propionic acid) were identified and shown to significantly inhibit PLA(2). X. nematophila culture broth contained these seven compounds, while P. temperata subsp. temperata culture broth contained three compounds (BZA, acetylated FGV, and cis-cyclo-PY). BZA was detected in the largest amount among these PLA(2) compounds in both bacterial culture broths. All seven bacterial metabolites also showed significant inhibitory activities against immune responses, such as phenoloxidase activity and hemocytic nodulation; BZA was the most potent. Finally, this study characterized these seven compounds for their insecticidal activities against the diamondback moth, Plutella xylostella. Even though these compounds showed relatively low toxicities to larvae, they significantly enhanced the pathogenicity of Bacillus thuringiensis. This study reports bacterial-origin PLA(2) inhibitors, which would be applicable for developing novel insecticides.     

Aromatic amino acid biosynthesis and production of related compounds from p-hydroxyphenylpyruvic acid by rumen bacteria,protozoa and their mixture

Summary. Aromatic amino acid biosynthesis and production of related compounds from p-hydroxyphenylpyruvic acid (HPY) by mixed rumen bacteria (B), protozoa (P), and their mixture (BP) in an in vitro system were quantitatively investigated. Microbial suspensions prepared from mature, fistulated goats fed Lucerne (Medicago sativa) cubes and a concentrate mixture were anaerobically incubated at 39°C for 12 h. Tyrosine (Tyr), phenylalanine (Phe), tryptophan (Trp) and other related compounds in both supernatants and hydrolyzates of all incubations were analyzed by HPLC. Large amounts of Tyr (27.0, 47.0 and 50.8% of disappeared HPY in B, P and BP, respectively) were produced from 1 mM HPY during a 12-h incubation period. The formation of Tyr in P was 1.8 and 1.6 times higher than those in B and BP, respectively. Appreciable amounts of Phe (3–12% of the disappeared HPY) and Trp (2–10% of the disappeared HPY) were also produced from HPY in B, P, and BP. Phe synthesis in B and P was almost similar but Trp synthesis in B was 1.8 times higher than that in P. The biosynthesis of both Phe and Trp from HPY in BP was higher than those in B plus P. A large amount of p-hydroxyphenylacetic acid (about 45% of the disappeared HPY) was produced from HPY in B which was 1.9 times higher than that in P. p-Hydroxybenzoic acid produced from HPY in P was 1.6 times higher than that in B. Considerable amounts of phenylpropionic acid, phenyllactic acid, and phenylpyruvic acid (2–6% of the disappeared HPY) were produced only in B. Received March 21, 2001 Accepted July 4, 2001     

3-Phenyllactic acid is converted to phenylacetic acid and induces auxin-responsive root growth in Arabidopsis plants

Many microorganisms have been reported to produce compounds that promote plant growth and are thought to be involved in the establishment and maintenance of symbiotic relationships. 3-Phenyllactic acid (PLA) produced by lactic acid bacteria was previously shown to promote root growth in adzuki cuttings. However, the mode of action of PLA as a root-promoting substance had not been clarified. The present study therefore investigated the relationship between PLA and auxin. PLA was found to inhibit primary root elongation and to increase lateral root density in wild-type Arabidopsis, but not in an auxin signaling mutant. In addition, PLA induced IAA19 promoter fused β-glucuronidase gene expression, suggesting that PLA exhibits auxin-like activity. The inability of PLA to promote degradation of Auxin/Indole-3-Acetic Acid protein in a yeast heterologous reconstitution system indicated that PLA may not a ligand of auxin receptor. Using of a synthetic PLA labeled with stable isotope showed that exogenously applied PLA was converted to phenylacetic acid (PAA), an endogenous auxin, in both adzuki and Arabidopsis. Taken together, these results suggest that exogenous PLA promotes auxin signaling by conversion to PAA, thereby regulating root growth in plants.     

Survival of silage lactic acid bacteria in the goat gastrointestinal tract as determined by denaturing gradient gel electrophoresis

Aims: To determine the survival rate of silage lactic acid bacteria (LAB) in the ruminant gastrointestinal tract. Methods and Results: Wilted Italian ryegrass (Lolium multiflorum Lam.) silage (containing 1·9 × 10⁶ CFU LAB g^?1) was fed ad libitum to three goats equipped with rumen cannulae. Silage was given alone or with concentrates at a 1 : 1 ratio on a dry matter basis. Rumen fluid was then obtained 2, 4 and 8 h after the morning feeding. Denaturing gradient gel electrophoresis was performed to compare LAB communities in silage, rumen fluid and faeces. The LAB detected in the wilted silage included Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus murinus and Lactobacillus sakei. Bands indicative of Lact. murinus were detected in either the rumen fluid or faeces, whereas the bands indicative of Lact. plantarum, Lact. brevis and Lact. sakei were not. Although the rumen fluid LAB counts and volatile fatty acid concentrations were higher in goats fed silage plus concentrates compared with those fed silage alone, the LAB communities themselves remained unaffected. Sampling times and goat‐to‐goat variations did not affect the LAB communities found in the rumen fluid. Conclusion: LAB communities found in the gut are not remarkably affected by the consumption of silage LAB, even when the silage is accompanied by concentrates that facilitate gut fermentation. Significance and Impact of the Study: Although silage can improve probiotic function, it may be difficult for silage LAB to survive the digestive process in the ruminant gastrointestinal tract.     

Mass spectrometric technique for the determination of N-phosphonoacetyl-l-aspartic acid in serum

N-Phosphonoacetyl-l-aspartic acid (PALA), a potent inhibitor of aspartic acid transcarbamylase, is now undergoing Phase I clinical trials. Initial experiments revealed that PALA is not metabolized to phosphonoacetic acid (PAA) in humans. Thus PALA may be quantified in serum after in vitro conversion to PAA. Serum is deproteinized with perchloric acid, lipid extracted with methylene chloride, hydrolyzed with 8 N hydrochloric acid at 100° for 3 h, and evaporated to dryness with nitrogen. The residue is silylated, and PAA is quantified by monitoring the

ions of the protonated molecular ions of trimethylsilyl derivatives of PAA and phosphonopropionic acid (internal standard) obtained in chemical ionization with methane. Limit of detection is 0.5 μM (150 ng/ml) PALA using 1 ml serum. PALA was given by continuous infusion to cancer patients at various doses. Maximum levels of PALA (50–500 μM range) were obtained at the end of infusion, followed in most cases by biexponential decay. Persistent residual PALA levels (5 μM for 48 h after infusion) correlated with increased toxicity.     

Effect of feeding precursors on phenylalanine ammonia-lyase activity and coumarin accumulation in leaves of <Emphasis Type="Italic">Matricaria chamomilla</Emphasis> L.

Four-week-old chamomile (Matricaria chamomilla) plants were exposed for 72 h to 0.01, 0.1 and 1 mM phenylalanine (Phe) or tyrosine (Tyr). Phe at all concentrations significantly increased phenylalanine ammonia-lyase (PAL) activity (by 30, 76 and 90%, respectively) as well as accumulation of coumarin-related compounds (herniarin and its precursors (Z)- and (E)-2-β-D-glucopyranosyloxy-4-methoxycinnamic acids). Free Phe content increased significantly at the highest dose tested. Lower Tyr concentrations (0.01 and 0.1 mM) significantly increased PAL activity and increased free Tyr content, however free Phe content decreased. This indicated that Tyr-mediated stimulation of PAL is coupled to Phe consumption. Notwithstanding, Tyr had no effect on coumarin accumulation. Therefore we speculate that in chamomile a regulation/signalling mechanism could be operating in the pathway leading to coumarin synthesis. The malondialdehyde accumulation, an usual marker of stress in plants, was not significantly changed by amino acid supplements, suggesting that membrane damage is not the signal causing coumarin accumulation. In parallel experiment we observed that neither lower (0.25 × full strength), nor higher (3 × full strength) nitrogen concentration of nutrient solution compared to normal (1 × full strength, 205 mg N l^-1) solution used for Phe/Tyr supply affected herniarin and GMCAs accumulation. This indicates that Phe had stimulatory effect on PAL activity and coumarin metabolism.     

Thin layer chromatographical detection of tyrosine produced from <Emphasis Type="SmallCaps">l</Emphasis>-[U-<Superscript>14</Superscript>C]phenylalanine by ruminal microbes

Summary.  Thin layer chromatographical detection of tyrosine (Tyr) synthesized from l-[U-¹⁴C]phenylalanine (Phe) (1 mM) by rumen bacteria (B) and protozoa (P) collected from fistulated Japanese Goat was carried out. About 16 and 12% of the added Phe was converted to Tyr by B and P, respectively. Large amount of radioactivity in ether fractions indicated an abundant production of aromatic acids from Phe. Small amount of radioactivity found in CO₂ fractions implied an occurrence of considerable decarboxylation reaction(s) by rumen bacteria and protozoa. Received July 18, 2001 Accepted December 3, 2001     

The effect of natural and synthetic auxins on the growth,metabolite content and antioxidant response of green alga Chlorella vulgaris (Trebouxiophyceae)

The effect of exogenously applied natural [indole-3-acetic acid (IAA), phenylacetic acid (PAA), indole-3-butyric acid (IBA)] and synthetic [1-naphthaleneacetic acid (NAA)] auxins on the growth and metabolism of green microalga Chlorella vulgaris was examined. Exogenous auxins acted in a concentration-dependent manner on algal growth. Phytohormones at concentration of 100 μM inhibited algal growth expressed as the number of cells. IAA and IBA displayed the highest biological activity at 0.1 μM, whereas PAA and NAA were characterized by the greatest stimulatory effect on the number of cells at 1 μM. Treatment with IAA and IBA at 0.1 μM or NAA and PAA at 1 μM increased the concentration of photosynthetic pigments, monosaccharides and soluble proteins in C. vulgaris. Moreover, all auxins stimulated enzymatic (ascorbate peroxidase, catalase, superoxide dismutase) and non-enzymatic antioxidant (ascorbate, glutathione) systems in C. vulgaris, and therefore, suppressed lipid peroxidation and hydrogen peroxide accumulation. The data supports the hypothesis that auxins play a central role in the regulation of C. vulgaris growth and metabolism and the components of cellular redox systems that are thought to have a prominent role in the regulation of auxin-dependent processes.     

Studies of fluorescent components of the “heparin” drug and its complexing with phenylalanine, tyrosine, and tryptophan

Impurities of free aromatic amino acids (Phe and Tyr) and the elastin protein were found in the heparin commercial drug (Hep) by spectral luminescent and spectrophotometric methods. The fluorescence quenching of the Trp, Tyr, and Phe amino acids by the Hep drug was studied, and the Stern-Folmer constants (K) that reflected stability of the Hep complexes with amino acids were determined. The stability of AA-Hep complexes increased in the following sequence: Trp < Tyr < Phe (K = 19 ± 2 < 39 ± 3 < 710 ± 70 M^?1, respectively). These values probably determined the dominant contribution of the phenylalanine impurity in the heparin drug. The contamination of animal elastin whose structure differed from that of the human elastin is thought to be a reason for allergic reactions and even anaphylactic shock during medical treatment with this drug.     

Engineering of substrate specificity of D-amino acid oxidase from the yeast Trigonopsis variabilis: Directed mutagenesis of Phe258 residue

Natural D-amino acid oxidases (DAAO) are not suitable for selective determination of D-amino acids due to their broad substrate specificity profiles. Analysis of the 3D-structure of the DAAO enzyme from the yeast Trigonopsis variabilis (TvDAAO) revealed the Phe258 residue located at the surface of the protein globule to be in the entrance to the active site. The Phe258 residue was mutated to Ala, Ser, and Tyr residues. The mutant TvDAAOs with amino acid substitutions Phe258Ala, Phe258Ser, and Phe258Tyr were purified to homogeneity and their thermal stability and substrate specificity were studied. These substitutions resulted in either slight stabilization (Phe258Tyr) or destabilization (Phe258Ser) of the enzyme. The change in half-inactivation periods was less than twofold. However, these substitutions caused dramatic changes in substrate specificity. Increasing the side chain size with the Phe258Tyr substitution decreased the kinetic parameters with all the D-amino acids studied. For the two other substitutions, the substrate specificity profiles narrowed. The catalytic efficiency increased only for D-Tyr, D-Phe, and D-Leu, and for all other D-amino acids this parameter dramatically decreased. The improvement of catalytic efficiency with D-Tyr, D-Phe, and D-Leu for TvDAAO Phe258Ala was 3.66-, 11.7-, and 1.5-fold, and for TvDAAO Phe258Ser it was 1.7-, 4.75-, and 6.61-fold, respectively.