Bone marrow mononuclear cells versus G-CSF-mobilized peripheral blood mononuclear cells for treatment of lower limb ASO: pooled analysis for long-term prognosis.

In this study, we report the comparative result of long-term clinical prognoses for patients with no-option critical limb ischemia (CLI) caused by arteriosclerosis obliterans, who are implanted with autologous bone marrow mononuclear cells (BMMNC; n=74) or G-CSF-mobilized (M)-PBMNC (n=111), as no information is available on how the two treatments compare in terms of long-term prognosis, such as survival or amputation. We performed pooled analysis using data from two previous cohort studies. All patients had disease of Fontaine classification III or IV. The endpoints were OS and amputation-free survival (AFS). After adjustment for history of dialysis and Fontaine classification, there was no significant difference between the two treatments with respect to OS (hazard ratio (HR)=1.49; 95% confidence interval (CI)=0.74-3.03, P=0.26) or AFS (HR=0.96; 95% CI=0.61-1.51, P=0.87). The negative prognostic factors affecting OS or AFS were the small number of CD34-positive cells collected, history of dialysis, Fontaine classification, male sex and older age. These results suggest that there was no significant difference in long-term prognosis between patients treated with BMMNC and those treated with M-PBMNC. The number of CD34-positive cells collected was an important prognostic factor for amputation and death.

Degradation of tryptophan and related indolic compounds by ruminal bacteria, protozoa and their mixture in vitro.

In vitro experiments were conducted to examine the degradation of d- and l-isomers of tryptophan (Trp) and 10 related indolic compounds by mixed rumen bacteria (B), protozoa (P) and a combination of the two (BP). The analyses were carried out by HPLC. d-Trp (1.0 mM) was not degraded by rumen microorganisms during the 24-h incubation period. The net degradation of 1 mM l-Trp was 46.5%, 8.7% and 80.0% by B, P and BP suspensions, respectively. Trp was degraded into indoleacetic acid, indolelactic acid and indole by rumen bacteria and protozoa, and into skatole, p-cresol and indolepropionic acid by rumen bacteria only. Of them, indoleacetic acid was the major product of Trp found in B (15.4%) and P (3.1%), and skatole in BP (43.2%). This is the first report of the production of indolelactic acid and p-cresol from Trp by rumen microbes. Starch, d-glucose, salinomycin and monensin inhibited the production of skatole and indole from Trp, and skatole from indoleacetic acid by rumen bacteria.

Studies on the utilization of methionine sulfoxide and methionine sulfone by rumen microorganisms in vitro.

An in vitro experiment was conducted to test the ability of mixed rumen bacteria (B), protozoa (P), and their mixture (BP) to utilize the oxidized forms of methionine (Met) e.g., methionine sulfoxide (MSO), methionine sulfone (MSO(2)). Rumen contents were collected from fistulated goats to prepare the microbial suspensions and were anaerobically incubated at 39 degrees C for 12 h with or without MSO (1 mM) or MSO(2) (1 mM) as a substrate. Met and other related compounds produced in both the supernatants and hydrolyzates of the incubation were analyzed by HPLC. During 6- and 12-h incubation periods, MSO disappeared by 28.3 and 42.0%, 0.0 and 0.0%, and 40.6 and 62.4% in B, P, and BP suspensions, respectively. Rumen bacteria and the mixture of rumen bacteria and protozoa were capable to reduce MSO to Met, and the production of Met from MSO in BP (156.6 and 196.1 micromol/g MN) was about 17.3 and 14.1% higher than that in B alone (133.5 and 171.9 micromol/g MN) during 6- and 12-h incubations, respectively. On the other hand, mixed rumen protozoa were unable to utilize MSO. Other metabolites produced from MSO were found to be MSO(2) and 2-aminobutyric acid (2AB) in B and BP. MSO(2) as a substrate remained without diminution in all-microbial suspensions. It was concluded that B, P, and BP cannot utilize MSO(2); but MSO can be utilized by B and BP for producing Met.

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

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 degrees 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.

Thin layer chromatographical detection of tyrosine produced from L-[U-(14)C]phenylalanine by ruminal microbes.

Thin layer chromatographical detection of tyrosine (Tyr) synthesized from L-[U-(14)C]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 CO(2) fractions implied an occurrence of considerable decarboxylation reaction(s) by rumen bacteria and protozoa.

Intracerebroventricular injection of pipecolic acid inhibits food intake and induces sleeping-like behaviors in the neonatal chick.

It has been demonstrated that L-pipecolic acid (L-PA), a major metabolic intermediate of L-Lysine (L-Lys) in the brain, is involved in the functioning of Gamma-aminobutyric acid. In the present work the effect of intracerebroventricular (i.c.v.) administration of L-PA, and its relatives, on food intake and behavior in neonatal chicks was investigated. The i.c.v. injection of 1 mg of L-PA and D-PA significantly inhibited food intake during the 2 h following injection, whereas greater than 2 mg of L-Lys was required to inhibit food intake. In behavioral tests, the i.c.v. injection of L-PA reduced active wakefulness and feeding behavior while inducing sleeping-like behavior in chicks. These results suggest that L-PA has an important role for the regulation of behaviors in the neonatal chick after conversion from L-Lys in the brain.

Biosynthesis of methionine from homocysteine, cystathionine and homoserine plus cysteine by mixed rumen microorganisms in vitro.

This study quantitatively investigated the biosynthesis of methionine (Met) and the production of related compounds from homocysteine (Hcys), cystathionine (Cysta), and homoserine (Hser) plus cysteine (Cys) by rumen bacteria (B) or protozoa (P) alone and by a mixture of these bacteria and protozoa (BP). Rumen contents were collected from fistulated goats to prepare the microbial suspensions and were anaerobically incubated at 39 degrees C for 12 h. Hcys, Cysta, and Hser plus Cys were catabolized by all rumen microbial fractions to different extents. B, P, and BP converted Hcys to Met with 2-aminobutyric acid (2AB) and methionine sulfoxide. The Met-producing ability of B (83.2 micromol g(-1) microbial nitrogen; MN) from Hcys was about 3.6 times higher than that of P in a 6-h incubation period. The ability of BP, during the same incubation period, was about 30.0% higher than that of B. Hcys, Met, and 2AB were formed when Cysta was incubated with B, P, or BP. Rumen microbial fermentation of Hser plus Cys led to the formation of Cysta, Met (through Hcys), and 2AB. Thus the results indicated that a trans-sulfurylation pathway for Met synthesis was operating in the rumen bacteria and protozoa. The results mentioned above have been demonstrated for the first time in B, P, and BP in the present study.

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 (150x4.6 mm I.D., 5 microm particle size) equipped with a guard column (4.0x4 mm, 5 microm 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 (microM) (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 microM, but Cit, Arg, Pro, and 4AB were not found, although all these four compounds were detected 1 h after feeding. Pro (390 microM) and 5AV (497.6 microM) were highest 1 h after feeding and then decreased. Orn levels before morning feeding were most similar to those after feeding.

Studies on the possibility of histidine biosynthesis from histodinol, imidazolepyruvic acid, imidazoleacetica acid, and imidazolelactic acid by mixed ruminal bacteria, protozoa, and their mixture in vitro.

The possibility of histidine (His) synthesis using a main biosynthetic pathway involving histidinol (HDL) and also the recycling capability of imidazolic compounds such as imidazolepyruvic acid (ImPA), imidazoleacetic acid (ImAA), and imidazolelactic acid (ImLA) to produce His were investigated using mixed ruminal bacteria (B), protozoa (P), and a mixture of both (BP) in an in vitro system. Rumen microorganisms were anaerobically incubated at 39 degrees C for 18 h with or without each substrate (2 mM) mentioned. His and other related compounds produced in both the supernatants and hydrolyzates of the incubation were analyzed by high-performance liquid chromatography. B, P, and BP suspensions failed to show His synthesizing ability when incubated with HDL. His was synthesized from ImPA by B, P, and BP. Expressed in units "per gram of microbial nitrogen (MN)", ImPA disappearance was greatest in B (72.7 micromol/g MN per hour), followed by BP (33.13 micromol/g MN per hour) and then P (18.6 micromol/g MN per hour) for the 18-h incubation period. The production of His from ImPA in B (240.0, 275.9, and 261.2 micromol/g MN in 6, 12, and 18 h incubation, respectively) was about 3.5 times higher than that in P (67.3, 83.8, and 72.7 micromol/g MN in 6, 12, and 18 h incubation, respectively). Other metabolites produced from ImPA were ImLA, ImAA, histamine (HTM), and urocanic acid (URA), found in all microbial suspensions. ImLA as a substrate remained without diminution in all microbial suspensions. Although ImAA was found to be degraded to a small extent (3.4-6.3%) only after 18 h incubation, neither His nor other metabolites were detected on the chromatograms. These results have been demonstrated for the first time in rumen microorganisms and suggest that His may be an essential amino acid for rumen microorganisms.

Suppression of tumourigenicity, and induction of differentiation of the canine mammary tumour cell line MCM-B2 by sodium phenylacetate.

The authors evaluated the cell growth inhibition, reduction of tumourigenicity, and differentiation-inducing effects of sodium phenylacetate (NaPA) on a canine mammary tumour cell line. Treatment of the canine mammary tumour cell line (MCM-B2) with NaPA lead to the arrest of cell growth. Sodium phenylacetate induced changes in the cells to non-malignant characteristics, as indicated by a reduction of colony formation in semi-solid agar and a decrease in tumour formation in athymic mice. Moreover, NaPA induced morphological changes from a spindle-shaped to an epithelial-like appearance, and significant accumulation of lipid droplets in the cytoplasm. Immunohistochemically, these treated cells reacted clearly with the antibody for keratin/cytokeratin. Sodium phenylacetate treatment increased the expression of the milk-specific genes alpha-lactalbumin and beta-casein. The results of this study warrant an evaluation of NaPA in a clinical trial to establish its possible value as adjunctive treatment of malignant canine mammary tumours.

Biosynthesis of threonine from homoserine by mixed rumen microorganisms: an in vitro study.

The biosynthesis of threonine (Thr) by using the main biosynthetic pathway involving homoserine (Hser) was quantitatively investigated by mixed rumen bacteria (B), protozoa (P), and their mixture (BP) in an in vitro system. Rumen contents were collected from fistulated goats to prepare the microbial suspensions and were incubated anaerobically at 39 degrees C for 12 h with or without Hser (2 mm) as a substrate. Thr and other related compounds produced in both the supernatants and hydrolysates of the incubation were analyzed by HPLC. During a 12-h incubation period, 84.2%, 58.1%, and 92.0% of Hser disappeared in B, P, and BP suspensions, respectively. Rumen bacteria and the mixture of rumen bacteria and protozoa were demonstrated for the first time to produce Thr from Hser, and the production of Thr from Hser in BP (371.9 and 297.2 micromol/g MN) (MN, microbial nitrogen) was about 13.0% and 9.1% higher than that in B alone (329.2 and 272.5 micromol/g MN) during 6- and 12-h incubations, respectively. On the other hand, mixed rumen protozoa were unable to synthesize Thr from Hser. Other metabolites produced from Hser were found to be glycine (Gly) and 2-aminobutyric acid (2AB) in B and BP. In P, Gly and 2AB were not found. The results mentioned above indicated the abilities of rumen bacteria and the mixture of rumen bacteria and protozoa to synthesize Thr de novo from Hser and appeared as first-time report.

In vitro catabolism of histidine by mixed rumen bacteria and protozoa.

An in vitro study was conducted to examine the metabolism of histidine (His) by mixed rumen bacteria (B), mixed rumen protozoa (P), and a combination of the two (BP). Rumen microorganisms were collected from fistulated goats fed with lucerne cubes (Medicago sativa) and a concentrate mixture twice a day. Microbial suspensions were anaerobically incubated with or without 2 mm each of His, or histamine (HTM), or 1 mm urocanic acid (URA) at 39 degrees C for 12 h. His and other related compounds in both supernatant and microbial hydrolysates were analyzed by HPLC. After 6- and 12-h incubations, the net degradation of His was 26.1% and 51.7% in B, 13.5% and 20.9% in P, and 21.7% and 46.0% in BP, respectively. The rate of the net degradation of His in B (98.0 micromol/g microbial nitrogen/h) was about 2.6 times higher than that of P during a 12-h incubation period. His was found to be degraded into urocanic acid (URA), imidazolelactic acid (ImLA), imidazoleacetic acid (ImAA), and histamine (HTM). Of these degraded His was mainly converted into URA in all microbial suspensions. The production of ImLA and ImAA was higher in B than in P suspensions, whereas the production of HTM was higher in P than in B suspensions. From these results, the existence of diverse catabolic routes of His in rumen microorganisms was indicated.

Catabolism of methionine and threonine in vitro by mixed ruminal bacteria and protozoa.

In vitro studies were conducted to examine the metabolism of methionine (Met) and threonine (Thr) using mixed ruminal bacteria (B), mixed ruminal protozoa (P), and a combination of these two (BP). Rumen microorganisms were collected from fistulated goats fed with lucerne cubes (Medicago sativa) and a concentrate mixture twice a day. Microbial suspensions were anaerobically incubated with or without 1 mM each of the substrates at 39 degrees C for 12h. Met, Thr and their related amino compounds in both the supernatants and microbial hydrolyzates of the incubation were analyzed by HPLC. Met was degraded by 58.7, 22.1, and 67.3% as a whole in B, P, and BP suspensions, respectively, during 12h incubation. In the case of Thr, these values were 67.3, 33.4, and 76.2% in B, P, and BP, respectively. Met was catabolized by all of the three microbial suspensions to methionine sulfoxide and 2-aminobutyric acid. Catabolism of Thr by B and BP resulted in the production of glycine and 2-aminobutyric acid, while P produced only 2-aminobutyric acid. From these results, the existence of diverse catabolic routes of Met and Thr in rumen microorganisms was indicated.

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 (150x4.6 mm I.D., 5 microm particle size) column. All analytes were detected at 265 nm with UV detection. The limits of detection (microM) (S/N ratio, 3:1) and quantification (microM) (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 (microM) 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.

Determination of histidine and related compounds in rumen fluid by liquid chromatography.

A liquid chromatographic procedure was developed for quantitative determination of histidine (His), histidinol (HDL), histamine (HTM), urocanic acid (URA), imidazolepyruvic acid (ImPA), imidazoleacetic acid (ImAA), and imidazolelactic acid (ImLA) in rumen fluid. The method is based on direct injection analysis by UV absorbance detection at 220 nm. The separation was performed under 2 different chromatographic conditions on a LiChrospher 100 NH2 column. In the first chromatographic system, the mobile phase used for isocratic elution was 67 mM potassium phosphate buffer (monobasic and dibasic) pH 6.45-90% acetonitrile in water (21 + 79); in the second system, an acetonitrile gradient in 63 mM potassium phosphate buffer (monobasic) pH 3.0, obtained by addition of 60 mM phosphoric acid, was used. Analyses of both systems were completed within 32 and 25 min, respectively. The limits of detection of these compounds were (microM): His, 2.8; HDL, 3.7; HTM, 4.0; URA, 0.75; ImPA, 4.7; ImAA, 1.2; and ImLA, 1.3. Recovery of these compounds added to rumen fluid was 97.4-103.0% within a 1-day study and 95.4-99.0% on different day studies. Detectable levels of His were found in the deproteinized rumen fluid of goats, with average concentrations of 16.10, 10.43, 11.14, and 13.62 microM in the rumen fluid collected before the morning feeding and 2, 4, and 6 h after feeding, respectively. HDL, HTM, URA, ImPA, ImAA, and ImLA were not detected in the rumen fluid before and after feeding. Trp, Phe, and Tyr were also identified in the rumen fluid, with average concentrations of 8.25, 29.04, and 12.6 microM, respectively, before the morning feeding.

Tryptophan biosynthesis and production of other related compounds from indole and L-serine by mixed ruminal bacteria, protozoa, and their mixture in vitro.

Tryptophan (Trp) biosynthesis and production of other related compounds from 1 mM each of indole (IND), L-serine (Ser), and IND plus Ser by mixed ruminal bacteria (B), protozoa (P), and their mixture (BP) in an in vitro system were quantitatively investigated. Ruminal microorganisms were anaerobically incubated at 39 degrees C for 12 h. Trp and other related compounds produced in both the supernatants and microbial hydrolyzates of the incubation were analyzed by HPLC. B, P, and BP suspensions were not able to produce Trp when incubated with only IND or Ser. Appreciable amounts of Trp (9.8, 3.1, and 6.6% of substrate) were produced from IND plus Ser after 12 h by B, P, and BP suspensions, respectively. Trp produced from IND + Ser in B was found only in the hydrolyzate, whereas it was found in the medium as a free form in P after a 12-h incubation period. Rumen bacteria and protozoa were separately demonstrated for the first time to produce Trp from IND plus Ser, and the ability of P to produce Trp from IND plus Ser was about one-third that of B in 12 h. Trp produced from IND plus Ser by B, P, and BP suspensions was simultaneously degraded into its related compounds, and, among them, indoleacetic acid (IAA) was a major product found in B. Production of IAA was 4.3, 0.3, and 3.2% of IND in 12 h by B, P, and BP suspensions, respectively. A small amount of skatole (SKT) (1.1 and 2.5% in B and BP, respectively) and p-cresol (CRL) (2.4 and 3.4% in B and BP, respectively) were also produced from IND plus Ser during 12-h incubation. P suspension produced no SKT or CRL from IND plus Ser in 12-h incubation. These results suggested for the first time that both rumen bacteria and protozoa have an ability to produce Trp from IND plus Ser, and the ability was higher in B than in P. The ratios of Trp produced from IND plus Ser to that from indolepyruvic acid by B, P, and BP were 1:3.4, 1:14.2, and 1:6.6 during 12-h incubation period. From these results, the degree of importance of producing Trp from IND plus Ser in the rumen was indicated.

Rapid determination of lysine in biological samples by isocratic liquid chromatography.

A simple, rapid, and sensitive method was developed for detection and quantitation of lysine (Lys) in various biological samples by isocratic liquid chromatography (LC). Samples containing Lys and other amino acids were derivatized with 9-fluorenylmethyl chloroformate (FMOC-CI). The mobile phase used for isocratic elution was 50 mmol/L sodium acetate buffer (pH 4.20)-acetonitrile (43 + 57, v/v). Lys was detected with a UV detector at 265 nm. The derivatized Lys eluted from a LiChrospher 100 RP-18 (150 x 4.0 mm id) column at a retention time of 5.6 min. The limit of detection was 0.73 mumol/L (signal-to-noise [S/N] ratio, 3:1), and the limit of quantitation was 2.37 mumol/L (S/N ratio, 10:1). Lys recoveries from fortified biological samples were > 97.5%. Average Lys contents found in rumen fluid samples collected before the morning feeding and at 2.0, 4.0, and 6.0 h after feeding were 4.26, 3.34, 3.58, and 3.82 mumol/L, respectively. The hydrolysate of a sample of mixed rumen microorganisms collected before the morning feeding was determined to contain 1.372 mumol/mg microbial nitrogen in the form of Lys. The Lys concentrations of human plasma, goat plasma, human urine, and goat urine were 140.0, 102.0, 58.0, and 32.0 mumol/L, respectively.

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 microM in rumen liquor and 0.01 and 0.05 microM 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 microM Pip and Lys), MB9 (5.0 microM Pip and Lys), plasma (7.1 microM Pip and 85.6 microM Lys) and rumen liquor (28.4 microM Pip and 10.2 microM 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 microM in rumen liquor, and 7.3+/-2.5 and 137.3+/-38.0 microM 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.

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

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 degrees C for 12 h with or without 1 mM of L-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 12 h incubation in B (183.6 mumol/g MN) was 4.3 times higher than that in P. One of the intermediate products between Phe and Tyr seems to be p-hydroxyphenylacetic acid. The rate of the net degradation of Phe incubation in B (76.0 mumol/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.

Cloning of cDNA encoding a novel mouse DNA topoisomerase III (Topo IIIbeta) possessing negatively supercoiled DNA relaxing activity, whose message is highly expressed in the testis.

We cloned cDNA encoding a novel mouse homologue of DNA topoisomerase III (mTOP3beta). The nucleotide sequence contains an open reading frame of 863 amino acids, and the deduced molecular mass of the coded protein is 96.9 kDa. The overall sequence of mTOP3beta has a 48 and 36% identity with mouse TOP3alpha at the nucleotide and amino acid level, respectively. DNA topoisomerase IIIbeta was expressed using a baculovirus expression system and purified. The purified DNA topoisomerase IIIbeta had activity to relax negatively supercoiled DNA. Relaxation of supercoiled DNA was partial at 37 degreesC and complete relaxation was observed at higher temperatures. mTOP3beta mRNA was strongly expressed in the testis and relatively strongly in the brain. The levels of TOP3beta mRNA in the testis increased slightly 14 days and considerably 17 days after birth, when the cells in the pachytene phase begin to appear and increase.