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Formation of lysine from stereoisomers (SI) of 2,6-diaminopimelic acid (DAP) and the epimerization between the three SI of DAP (DAP-SI) by rumen protozoa and bacteria were examined. Mixed rumen protozoa (P) and bacteria (B) were isolated from the rumen of goats given a concentrate and hay cubes and incubated separately with and without a mixture and a single one of the three DAP-SI. In P suspensions, mixed DAP-SI decreased by 10.59% as a whole and converted mainly to lysine by 8.41% during 12 h incubation. When meso-, L- and D-DAP were added singly to the media, the results showed that each DAP-SI interconverted and produced lysine. This means that mixed rumen protozoa have an ability to synthesize lysine from not only meso-DAP but also from D- and L-DAP, though probably via meso-DAP, and hence have DAP epimerase activities for the reversal conversion of each DAP-SI. This is the first discovery to show the interconversion of DAP-SI and synthesis of lysine from them by protozoa. In B suspensions, mixed DAP-SI decreased by 10.92% as a whole and converted to lysine by 4.20% during 12 h incubation. When a single DAP-SI was added to the media, meso-, L- and D-DAP were interconverted and then converted to lysine by the rumen bacteria as well as the protozoa. This also means that mixed rumen bacteria have DAP epimerase activities to interconvert DAP-SI and have an ability to synthesize lysine from not only meso-DAP but also from L- and D-DAP, and this is also the first finding in rumen bacteria.

International audience; The degree of autolysis and presence of cell-wall degrading enzymes in an anaerobic ruminal fungus, Piromyces communis OTSI, grown in liquid medium, was monitored to evaluate the effect of self-digestion on fungal biomass. After a 30 days incubation period fungal dry weight decreased by 45% and the cell wall component chitin decreased by 22%. Chitinase activity detected in the supernatant was mainly of the endotype and peaked at day 6 of the incubation. 棺-1, 3-glucanase was detected from day 4 and increased throughout the incubation period. Autolysis was a slow process, and under natural conditions it is unlikely that it plays a significant role in the degradation of the spent fungal vestative stage in the rumen.

Seventeen ciliate species were identified from water buffalo and Kedah Kelantan cattle in Malaysia. Although most of these species have been detected from domestic ruminants in tropical and temperate areas, the presence of Metadinium ypsilon and Ostracodinium trivesiculatum is characteristic of Malaysian cattle and water buffalo. The average numbers of ciliates per milliliter of rumen contents were similar to those found in other areas. However, the level of Metadinium and Eudiplodinium , considered to be fermenters of cellulosic materials, was higher. This suggests that Malaysian cattle and water buffalo possess a ciliate protozoal composition favorable for digestion of cellulosic feed stuffs. There were no differences in the ciliate profile between the cattle and water buffalo. A similar pattern of diurnal change in the ciliate number of respective genera to that reported previously was also found in both Malaysian cattle and water buffalo.

A rapid method for the determination of phenylalanine (Phe), tyrosine (Tyr), benzoic acid (BZA), phenylacetic acid (PAA), phenyllactic acid (PLA), phenylpyruvic acid (PPY), phenylpropionic acid (PPR), and cinnamic acid (CNM) in goat rumen fluid was established by high-performance liquid chromatography (HPLC). The mobile phase used for isocratic elution was methanol-sodium acetate buffer (pH 6.5) (8:92, v/v). The compounds were monitored at 220 nm with a UV detector. A 5-microliters portion of the filtrated rumen fluid was analyzed and the analysis was completed within 20 min. The minimum detectable limits (microM) of these compounds were: 12 for Phe, 3 for Tyr, 3 for BZA, 9 for PAA, 12 for PLA, 15 for PPY, 20 for PPR, and 8 for CNM. The average contents of Phe, BZA, PAA, PLA, and PPR in the rumen fluid of three goats were 15.4, 73.7, 615.9, 51.1, and 39.9 microM before morning feeding, 17.0, 123.7, 650.4, 208.2, and 502.4 microM at 3 h after feeding, and 18.4, 124.2, 510.0, 129.9, and 178.5 microM at 6 h after feeding, respectively. Of these compounds PAA was present at the highest concentration both before and after feeding. The content of PPR extremely increased especially at 3 h after feeding. The other three compounds, i.e. Tyr, PPY, and CNM, were not detected in goat rumen fluid.

This paper investigates a estimation method of angular velocities and attitude angles from acceleration signals. Our sensor system measures 6-DOF motions by resolving multiple acceleration signals. In this estimation, we combine the motion characteristic with a Kalman filter. Our method generates a required observational signal, and non-periodically corrects to the observational signal using detected translational accelerations with gravitational components. Finally, we investigated the proposed method in an experiment involving vehicle motion, and demonstrate that our sensor system and the proposed method perform well.

Mixed rumen ciliate protozoa (mainly Entodiniinae) from goats have two kinds of protease; one has a pH optimum of 3.0, the other is active at neutral or alkaline pH. The protease active at neutral or alkaline pH was partially purified from the supernatant after centrifugation of sonicated mixed rumen ciliate protozoa. The supernatant was chromatographed on Bio-Gel A-1.5m and a partially purified protease was obtained. This protease had a molecular weight of more than 400,000. When the sonicated protozoa were heated at 55°C for 15min, the active peak from the Bio-Gel A-1.5m column was shifted to a lower molecular weight, 27,000. The high molecular weight protease was strongly activated by high temprature and SDS, and inhibited by E-64 c. The protease degraded many proteins including those found in rumen bacteria. These findings suggest that rumen ciliate protozoa have high molecular weight protease that plays a role in the digestion of feed and bacterial protein.

The peptidoglycan component of most bacterial cell walls contains the amino acid, 2,6-diaminopimelic acid (DAP), which can exist in three stereoisomeric forms. A chiral ligand-exchange HPLC method is described that is capable of separating mixtures of these isomers without derivatisation so that they may be used as substrates in subsequent biological experiments. The conditions for separation were optimised so that DAP was eluted from the chiral column, MCI gel CRS1Ow, with a mobile phase of 2 mM CuSO4-methanol (98:2, v/v) at 40°C. The elution times for the dd -, meso- and ll -isomers were 10.5, 14.6 and 34.7 min, respectively, as confirmed by circular dichroism.

This paper describes a calibration method of 6-DOF acceleration sensor system using dual-axis type linear accelerometers. In our sensor, 6-DOF acceleration can be resolved using the geometrical condition of the accelerometers. Therefore, the measurement error results from the alignment error of the accelerometers. The error analysis shows to be able to identify the error only using some limited linear accelerations. Moreover, in dual-axis type accelerometers, there exit some dependency among each axis-sensitivities. Then, we a new calibration method to above case, and investigate the influence of the error margin included in the calibration. According to the analysis, it is shown that the geometrical alignment error to be identified, which absolute value is small or near zero in comparison with other, can be assumed zero. As a result, more efficient calibration is made possible by decreasing the number of calibration data needed in the calibration. Finally, the availability of the proposed calibration method and our sensor system are verified through vehicle motion experiments. The results show that it has good performance for a complex motion.

ルーメン微生物によるタンパク質の分解機構を知るために,プロトゾアのエキソペプチダーゼ活性について検討した.全エキソペプチダーゼ活性はカゼインを基質として培養後,あるいは酵素反応後の遊離したアミノ基とペプチド量の差から求めた.山羊ルーメンから得た混合プロトゾアを塩類緩衝液中で培養したところ,活性はプロトゾアの細胞内画分に認められた.この活性は超音波破壊したプロトゾア細胞の遠心沈殿に主に分布していたことから,エキソペプチダーゼが細胞膜などに結合したものであることが考えられた.最適pHを超音波破壊したプロトゾアについて検討したところ,pH7.3付近にピークが認められたが,それより低いpHでもさらに高い活性が認められた.一方、ロイシル-β-ナフチルアミドを基質としてロイシンアミノペプチダーゼ様活性も測定したところ,細胞内の分布では可溶性画分に存在し,最適pHは7.8であった.これらのことからプロトゾアのエキソペプチダーゼには多くの種類があるものと考えられた.全エキソペプチダーゼ活性,ロイシンアミノペプチダーゼ様活性ともにアミノペプチダーゼBとロイシンアミノペプチダーゼの特異的阻害剤であるウベニメクス(ベスタチン)ではわずかに阻害されたにすぎなかった.したがって,プロトゾアにおいてはアミノペプチダーゼの関与は少ないことが示唆された。

In vitroでのルーメンプロトゾア(P懸濁液),ルーメンバクテリア(B懸濁液)およびそれらの混合懸濁液(BP懸濁液)によるL-トリプトファン(Trp)からのインドール(Ind)およびスカトール(Skt)の生成を確認するとともに,これらの微生物懸濁液によるTrpからのIndおよびSkt生成に及ぼすサリノマイシン(SL)の影響を検討する目的で本研究を計画した.また,同時に,IndおよびSktがプロトゾアの生存および上記の各種微生物懸濁液中における揮発性脂肪酸(VFA)の生成に及ぼす影響も検討した.ルーメン微生物は,フィスチュラ装着山羊から採取した.その結果,P懸濁液は,12時間の培養中にTrpからIndのみを生成した.SLはP懸濁液におけるIndの生成を促進した.BおよびBP懸濁液中では,ともにTrpからSktおよびIndが生成された.そして,これらの生成はSLによりほぼ完全に抑制された.低濃度(2mM)のIndおよびSkt存在下では,BP懸濁液申のプロトゾアは長時間生存した.そして,BおよびBP懸濁液中における総VFAの生成を促進し,また,BP懸濁液中におけるプロピオン酸と酪酸のモル百分率を増加させた.

本実験は,牛の肝臓のMSOレダクターゼを精製するにとを目的とした.精製にはpH処理,疎水クロマトグラフィー,陰イオンクロマトグラフィー,ヒドロキシアパタイトクロマトグラフィーを用いた.精製された酵素は,ポリアクリルアミド電気泳動で,バンドが1本であることを確認した.また,同時にチオレドキシンおよびチオレドキシンレダクターゼも部分精製した.精製MSOレダクターゼは,ジチオトレイトールの存在下ではMSOをメチオニンに還元するが,NADPHでは還元が起こらなかった.しかし,チオレドキシンおよびチオレドキシンレダクターゼを加えるとNADPHで高いMSO還元活性が得られた.このことから,大腸菌や酵母などと同様に,牛の肝臓のMSOレダクターゼもチオレドキシン系が必要であると考えられた.

A simple, rapid and sensitive assay method for plasma N tau-methylhistidine by isocratic high-performance liquid chromatography has been developed. The deproteinized plasma was treated with o-phthalaldehyde-2-mercaptoethanol. The derivatives were separated on a LiChrospher 100 RP-18 column within 10 min. The detection limit for N tau-methylhistidine was 0.5 pmol. The plasma N tau-methylhistidine content of beef cattle and dairy cows was 0.038 +/- 0.004 and 0.017 +/- 0.002 nmol/ml/kg, respectively.

リン酸緩衝液(MB 9)およびザイッ〓過ルーメン液(SFRL)を培地として,ルーメン繊毛虫類のうち,特に,貧毛類+全毛類懸濁液並びに貧毛類単独懸濁液による溶液状カゼインの凝固と分解を検討した.SFRLを用いて4時間インキュベートした場合,貧毛類+全毛類懸濁液中では,溶液状カゼインは凝固し,これが分解されたが,貧毛類単独懸濁液中では,凝固せず,分解も起こらなかった.これらの結果から,貧毛類には溶液状カゼインをそのままの状態で分解する能力はないと考えられた.全毛類は,SFRL中でも溶液状カゼインを凝固させるが,全毛類の凝固カゼイン分解力はそれほど大きいとは考えられなかった.したがって,凝固カゼインは,主に,貧毛類によって体内に取り込まれた後に分解されると考えられた.このように,SFRL中での溶液状カゼインの分解においては,貧毛類と全毛類との間の共生的協同作用の存在が認められた.全毛類はカゼイン凝固物質を分泌し,その活性はSFRL中でも阻害されないと考えられた.貧毛類もこれを分泌するが,その活性はSFRL中に存在するある種の物質により阻害されると考えられた.関連して,カゼイン凝固物質とそのプロテアーゼ活性について考察した.

必須アミノ酸(ラット)のうち遊離の分枝アミノ酸(BCAA),フェニルアラニンおよびヒスチジンについては,ルーメン繊毛虫類による代謝様式がまだ知られていない.そこで,本研究では分枝アミノ酸を取り上げ,ルーメン繊毛虫類によるそれらの代謝を検討した.ヘイキェーブおよび濃厚試料を給与している山羊(日本在来種)のルーメン内容物から常法により繊毛虫類を採取し,洗浄後これを絶食条件下の抗生物質を含む緩衝液(MB9)中で,39°C,6時間インキェベーションを行なって細菌の影響を除去した後,再洗浄を行ない,14C標識BCAA並びに抗生物質を加えたMB9中で.さらに12時間インキェベーションを行なった.インキェベーション用容器としては,中心井付きのワールブルグフラスコを用いた.インキュベーション前後に分析用試料を採取し,遠心上清液および虫体加水分解物中のアミノ酸および遠心上清液中の揮発性脂肪酸(VFA)を定量した,さらに,それらの放射能並びにCO2画分の放射能を液体シンチレーションカウンターで測定した.その結果,絶食繊毛虫類は,放射性のBCAAからCO2およびエーテル可溶物質,特に,C4およびC5のVFAを生成していることが分かった.本研究ではそれらのVFAを同定できなかったが,過去のルーメン細菌などの結果から,C4-VFAはイソ酪酸でバリンから生成されると考えられた.また,C5-VFAには,少なくともイソ吉草酸および2-メチル酪酸が含まれ,それぞれ,ロイシンおよびイソロイシンから生成されると考えられた.さらに,BCAAの分解速度についても考察した.

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.

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.

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.

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 Γ-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.

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.

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.

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.

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 (r0.999). 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.

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.

A lambda phage cDNA expression library was constructed from washed suspensions of the rumen ciliate protozoan, Entodinium caudatum, which had been maintained in an isolated, monofaunated sheep. The library was screened using an anti-E. caudatum antiserum raised in rabbits against sonically disrupted protozoa, DNA sequences for two centrins or caltractins, a subfamily of the EF-hand Ca(2+)-modulated proteins which are closely related, highly conserved cytoskeletal proteins, were identified and characterised. Their phylogenetic position was established relative to other centrin gene sequences. The two proteins showed homology to Paramecium tetraurelia centrins: 50 and 52% identities and 71 and 75% similarities in the protein sequence, over 99 and 110 amino acids fragments. Codon usage and indices revealed the E. caudatum follows universal codon usage, but with a restricted number of codons, and has a low GC content.

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 degrees C and compounds were monitored at 215 nm with a UV absorbance detector after injection of 10 microl of filtered rumen fluid. Analysis was completed within 40 min. The minimum detectable limits of quantification (microM) 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.

International audience; A membrane-bound chitinase from cell wall fractions of the anaerobic ruminal fungus, Piromyces communis OTS1, was purified by affinity chromatography, gel filtration, and chromatofocusing. The molecular size of the chitinase was estimated by gel filtration to be 42.4 kDa and by SDS-PAGE to be 44.8 kDa, and its pI was 4.4. Activity was inhibited by Hg2+ and allosamidin. The activity at 39 degrees C was greatest at pH 6.0. It had an 'endo' type action. Solubilization tests indicated that plasmalemma-bound chitinase was held in place by an electrostatic type interaction. Characterization of the membrane-bound chitinase was more similar to that of extracellular chitinase than cytosolic chitinase. This suggested that membrane-bound chitinase was the origin of extracellular chitinase.

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.

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.

New high-performance liquid chromatographies were examined and applied for the analyses of the total (with one peak) and separate three stereoisomers of 2,6-diaminopimelic acid (DAP) in hydrolysed mixed rumen bacteria. The methods start with the reaction of DAP with 1-fluoro-2,4-dinitrophenylalanine amide for derivatisation. A mixture of 0.05 M triethylamine phosphate (pH 3.0) and acetonitrile (72:28, v/v) was used as an isocratic mobile phase for the total DAP determination (method 1), and a mixture of both solutions (78.5:21.5, v/v) for the determination of the separate three stereoisomers of DAP (method 2). The flow-rate was 1 ml/min; column, Merck LiChrospher 100 RP-18 (250 x 4 mm I.D.) of 5 microns particle size; column temperature, 40 degrees C; wavelength of detector, 325 nm. The retention times were 17.5 min for total DAP (method 1), and 39.4, 63.1 and 77.7 for meso-, LL- and DD-DAP, respectively (method 2). Lysine can also be determined by method 2 and the retention time was 122.8 min. The minimum detectable limit was 2.5 microM. The average analytical recoveries were 98.6% for total DAP and 99.1%, 100.4% and 100.2% for meso-, LL- and DD-DAP, respectively. The content of total DAP of the hydrolysed rumen bacteria collected from three goats fed lucerne cube and concentrate ranged from 25.55 to 27.36 mumol/g bacterial DM (method 1). The contents of the separate stereoisomers of DAP in the hydrolysed rumen bacteria from the three goats ranged from 19.64 to 22.06 and from 4.98 to 5.21 mumol/g bacterial DM for meso- and LL-DAP, respectively (method 2). DD-DAP was not detected.

International audience; Homogenates from a mixed ruminal protozoal population and a ruminal protozoon Entodinium caudatum were analyzed for chitinolytic and lysozyme activities by sodium dodecyl sulfate polyacrylamide gel electrophoresis. For chitinase activity, up to eight bands in mixed protozoa and seven bands in E. caudatum were detected. Estimated molecular mass ranged from 70 to 110 kDa. These enzymes did not display lysozyme activity. N-Acetyl-beta-glucosaminidase activity was also detected in both samples with an estimated molecular mass of 37 kDa. Lysozyme activity in mixed protozoa was present in two major and three minor bands, where one major band displayed the same motility as chicken egg white (CEW) lysozyme, and the other had an approximate molecular mass of 17.5 kDa. The latter remained active even when denatured in the presence of dithiothreitol and renatured under anaerobic conditions. Entodinium caudatum presented one major band coincident with that of CEW lysozyme and a minor band at the 17.5-kDa point. This study showed that protozoal chitinase and lysozyme activities are originated from several enzymes and that none of these enzymes exhibited both activities.

International audience; The cryopreservation of an anaerobic rumen fungus, Piromyces communis OTS1, was examined at -84 degrees C using dimethyl sulfoxide, propylene glycol or ethylene glycol as cryoprotectants. Ethylene glycol was the most effective agent, combining high survival and low toxicity, followed by dimethyl sulfoxide and propylene glycol. Cell-free rumen fluid in the cryopreservation medium decreased the toxicity of the cryoprotectant agents and also had a protective action per se. A survival of 80% after 1 year storage was obtained when samples with an initial zoospore density of 5 x 10(4) zoospores/ml were equilibrated for 15 min in medium containing 0.64 M ethylene glycol and 5% cell-free rumen fluid, then frozen with dry ice and stored at -84 degrees C.

Isotricha jalaludinii n. sp. found in the rumen of lesser mouse deer, Tragulus javanicus, in Malaysia was described and illustrated. This new species is characterized by the location and direction of the vestibulum, shape of the macronucleus, and absence of a dent at the vestibular opening. The presence of single peculiar isotrichid species in the rumen of mouse deer, which is recognized as one of the most primitive ruminants, suggests that the isotrichid ciliates similar to I. jalaludinii and Isotricha intestinalis were established at a fairly early period during the evolution of ruminants.

International audience; Ruminal bacteria and protozoa, and cell-free rumen fluid, were tested for the presence of enzymes involved in the degradation of the fungal cell wall. Protozoal homogenate obtained by ultrasonication showed chitinase (EC 3.2.1.14) and N-acetyl-beta-glucosaminidase (EC 3.2.1.52) activities when assayed with fluorogenic 4-methylumbelliferyl substrates. The chitinase activity was predominantly of the 'exo'-type. Lysozyme (EC 3.2.1.17) and 1,3-beta-glucanase (EC 3.2.1.39) activities were also present in this fraction. All these activities, except lysozyme activity, were recovered mainly in the supernatant fraction of the homogenate (approximately 85% of the total activity). Lysozyme showed the same amount of activity in the precipitate and supernatant fractions. Bacterial homogenates had N-acetyl-beta-glucosaminidase activity in both supernatant and precipitate fractions. The specific activity was one-third that of the protozoa. Bacteria able to grow in a medium with chitin as the sole carbon source were recognized and counted. Cell-free rumen fluid was unable to degrade any of the substrates tested.

International audience; An anaerobic rumen fungus, Piromyces sp. strain OTS1, was incubated in the presence or absence of a mixed, A-type, protozoal population obtained from a goat, in a medium containing filter paper cellulose as energy source and antibiotics to suppress bacterial growth. Fermentation end products, cellulose degradation, and chitin as an indicator of fungal biomass were examined. In the presence of protozoa, total volatile fatty acids, notably propionate and butyrate, increased, and lactate decreased. In fungus-protozoan coincubations, formate was not detected at the end of the experiment and the amount of reducing sugars remained low throughout the incubation period. The fungal growth in the coincubations was negatively affected. While protozoal predation on zoospores was one mechanism of inhibition, mature fungal cells were also affected. Total cellulose degradation was greater in fungal monocultures, but the amount of cellulose degraded per unit of fungal biomass was 25% larger in the coincubations. The negative effects that the protozoal predatory activity had on the fungal growth and subsequently on the amount of cellulose degraded by Piromyces sp. strain OTS1 were partially attenuated by the protozoal fibrolytic activity or by an enhanced fungal activity due to a more favorable environment.

In vitroの系によりルーメンプロトゾアによる嫌気性ルーメン真菌の捕食を検討した.山羊から2種類の真菌,Neocallimastix sp.とPiromyces sp.を単離した.山羊から採取した混合ルーメンプロトゾアをMB9緩衝液に懸濁し,これに凍結乾燥真菌を加えて,嫌気的条件で39°C,24hまでインキュベートした.そして,培養液中の総キチン量と培養液中に遊離したグルコサミンおよびアミノ酸を経時的に測定した.Pirornycesの場合は有意ではなかったけれどもインキュベーション中に総キチン量が減少した.また,Neocallimastixではほんの少し減少する傾向が見られた.24h後に培地中に遊離したグルコサミンは両者とも有意に増加した(P〈0.05).したがって,ルーメンプロトゾアはキチン分解活性を持つと考えられた.この点は初めての証明である.プロトゾアが培地中に放出する遊離アミノ酸は真菌の存在により増加した(P〈〈0.01)ので,ルーメンプロトゾアは真菌タンパク質を代謝していると推察された.

This article briefly summarizes the metabolism of methionine and lysine in the rumen of ruminant animals and known and inferable effects of their metabolites on the nutrition and physiology of ruminant animals.

Entodinium species are important in catabolic protein metabolism by the mixed ruminal microbial population. This study was conducted to purify, and investigate properties of one of the enzymes involved in amino acid metabolism by Entodinium caudatum, glutamate-phenylpyruvate aminotransferase (GPA; EC 2.6.1.64). GPA was purified 74-fold from a cell-free extract by ammonium sulfate precipitation and column chromatography with phenyl-superose, DEAE-Toyopearl 650M, Sephacryl S-100 HR, and Sephadex G-100. The molecular mass of GPA was estimated by SDS–PAGE to be 65.0 kDa. The optimum pH was 6.0 and it was found to be reactive over a wide range of pH from 5.0 to 10.5. Maximum activity of GPA occurred at 45°C and the activity declined at temperatures over 55°C. GPA was stable below 60°C. Aminooxyacetate and phenylhydrazine were highly inhibitory, and SDS, EDTA, and some heavy metal ions also inhibited activity. The purification and characterisation of the enzyme will help to isolate the gene and ultimately to understand the role of GPA in both anabolic and catabolic amino acid metabolism by Entodinium caudatum.

Cell walls containing α,e-diaminopimelate-l,7-14C (DAP) was prepared from Escherichia coli isolated from the rumen. After incubation of ciliates with the cell walls, 22.0% of DAP contained in cell walls of E. coli was converted to lysine and pipecolate. Heat-treated mixed rumen bacteria and heat-treated cell walls of mixed rumen bacteria added to the culture medium of rumen ciliates increased 0.572 and 0.934 μmole/ml of sum of lysine and pipecolate, respectively.From these results, it is clear that rumen ciliate protozoa can form lysine from DAP contained in the mucopeptide of bacterial cell walls. One of the nutritional significance of inhabitation of ciliates in the rumen was revealed.

本研究では,混合ルーメン繊毛虫類から全毛類を完全除去する方法の改良を行なった.山羊から得た混合ルーメン繊毛虫類懸濁液を種々の濃度のD-マンノース溶液(緩衝液中に6%(w/v)まで)中で39°Cにてインキュベートした.その結果,4% (w/v) D-マンノース溶液(約0.250Mショ糖液に等張)で1時間処理をし,続いて,この溶液に,さらに,2% (w/v)分のD-マンノースを追加した溶液中で2時間処理を続けると,混合ルーメン繊毛虫類から全毛類が完全に除去されることが示された.本研究では,全毛類の密度は,最高時で,約40×103/ml (Isotricha: 25×103/ml: Dasytricha: 15×103/ml)であった.本研究により,全毛類の除去に要する時間を以前よりも3時間節約することができた.また,Isotrichaは,Dasytrichaよりも速くD-マンノースを摂取する傾向が認められた.

Metabolites arising from the catabolism of lysine by the mixed rumen bacteria were chromatographically examined by using radioactive lysine. After 6 hr incubation, 241 nmole/ ml of lysine was decomposed to give ether-soluble substances and CO2 by the bacteria and 90 nmole/ml of lysine was incorporated unchanged into the bacteria. δ-Aminovalerate, cadaverine or pipecolate did not seem to be produced from lysine even after incubation of the bacteria with addition of those three amino compounds to trap besides lysine and radioactive lysine. Most of the ether-soluble substances produced from radioactive lysine was volatile fatty acids (VFAs). Fractionation of VFAs revealed that the peaks of butyric and acetic acids coincided with the strong radioactive peaks. Small amounts of radioactivities were detected in propionic acid peak and a peak assumed to be caproic acid. The rumen bacteria appeared to decompose much larger amounts of lysine than the rumen ciliate protozoa did.