Takashi Nagasawa's research while affiliated with Iwate University and other places

Publications (14)

Article
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 chromatogr...
Article
本研究は,トリプトファン(Trp),インドールピルビン酸(IPA)およびインドール酢酸(IAA)を基質として,ルーメン細菌(B系),繊毛虫(P系)およびそれらの共存系(BP系)によるTrp,スカトール(Skt)および関連化合物の生成におよぼすサリノマイシン(SL)(5μg/ml)の影響を検討することを目的とした.ルーメン微生物は,1日2回アルファルファキューブと濃厚飼料を給与しているフィスチュラ装着山羊から採取した.12時間培養後,SL無添加のBP系およびB系におけるIPAからの遊離のTrp生成量は,それぞれ,0.7および2.2%と低い値であったが,SLを添加すると,それぞれ,58.9および16.4倍に増加した.P系におけるIPAからの遊離Trpの生成量はSL無添加でも35.3%と高かったが...
Article
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 biologic...
Article
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...
Article
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...
Article
A simple, rapid and sensitive method for the determination of plasma taurine by high-performance liquid chromatography in the isocratic mode has been developed. The deproteinized plasma was treated with fluorescamine. These derivatives were separated on a LiChrospher 100 RP-8 column within 15 min. The detection limit for taurine was 0.2 microM. The...
Article
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.
Article
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...
Article
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-methylhistidin...

Citations

... Rumen protozoa, besides contributing nutrients 3 to the host animal, help in digesting carbohydrate and protein containing feedstuff by secretion of various saccharolytic and proteolytic enzymes 4,5 in microbial protein turnover 6 , modulation of bacterial populations [7][8][9] and association with methanogens 8,10 . Numerous species of ciliates occupy the rumen, where they constitute about half of the total microbial biomass 11 . ...
... Investigation of tryptophan metabolism by mixed rumen protozoa, bacteria and their combination has enabled the identification of the relative contribution of these micro organisms to the production of skatole.Mohammed et al. (2003) andOkuuchi et al. (1993) have demonstrated that skatole is fo rmed solely by rumen bacteria and not by protozoa, however the interaction of the two micro-organisms in mixed cultures has relevance to the in vivo production of skatole from tryptophan in grazing ruminants.Okuuchi et al. (1993) fo und that rumen protozoa have an excellent ability to transaminate indolepyruvic acid to yield microbial tryptophan thus stimulating the bacterial production of indoleacetic acid and skatole.Mohammed et al. (2003) reported that 43% of tryptophan was converted to skatole in mixed bacterial and protozoal cultures after 24 hours of incubation. This is close to the value of 39% conversion reported byYokoyama and Carlson (1974) during the 24 hour incubation of radio labelled tryptophan. ...
... Deamination determines the rate of ruminal amino acid degradation (Annison et al., 1959;Lewis, 1962). SL has been shown to decrease amino acid breakdown (Hoshino et al., 1992;Okuuchi et al., 1993), and this may be due to the inhibitory effect of deamination as has been observed with other ionophores (Hino and Russell, 1985;Newbold et al., 1990;Russell and Martin, 1984;Van Nevel and Demeyer, 1977;Wallace et al., 1981). In the case of tryptophan metabolism, Okuuchi et al. (1993) reported that tryptophan production from indolepyruvic acid in all the rumen bacterial and protozoal suspensions was greatly enhanced by SL, but skatole and indole production from tryptophan by SL were completely inhibited in the rumen bacterial suspensions. ...
... The most common interaction between protozoa and the other groups is predation (Lee et al., 2001). However, comprehensive studies of the relationship between protozoa and fungi are limited to only a few publications (Morgavi et al., 1993(Morgavi et al., , 1994aMiltko et al., 2014). The predation of fungi by protozoa is a two-step process during which zoospores are engulfed and digested by ciliates. ...
... The lowering protozoal counts in ruminal fluid could be due to the highest NH 3 -N concentration, which has been reported by Kanjanapruthipong and Leng [20]. Protozoa did not utilize urea as a N source and amino acids are needed for protozoal synthesis [21]. This might potentially decrease protozoa synthesis in the rumen [20]. ...
... If Pl MeHis is an intermediate between myofibrillar protein-bound MeHis and Ur MeHis, the presents results suggest that Pl MeHis concentration may be used to estimate the degradation rate of muscle proteins in cattle. This agrees with Nagasawa et al. (1993) who demonstrated with feed deprived goats that Pl MeHis, along with Ur MeHis, is a valid index of muscle proteolysis in this species. In conclusion, the results reported in the present study indicate that 1 ) in twice-daily-fed cows, the energy deficit occurring during underfeeding and the subsequent endogenous protein mobilization are larger in energy-restricted than in nitrogen-restricted cows; 2 ) whatever the nitrogenous and(or) the energetic supplies of the diet, the time from the last meal is the main trigger for within-day changes in metabolite and hormone concentrations, the energy deficit becoming large 16 h after the beginning of the last meal; 3 ) during underfeeding, the nocturnal interprandial period, and short-term feed deprivation, VFA supplies from the rumen are reduced, and twicedaily-fed cows adapt to the three situations in the same manner by reducing the Ins:GH ratio, resulting in an enhanced body protein mobilization for oxidation and gluconeogenesis purposes. ...
... Moreover, Broderick et al. (1988) indicated that free peptides originating from dietary protein degradation might accumulate in the rumen and be available for protozoal attack, which might contribute to free TAA accumulation in the rumen. In the presence of RP because of active protozoal proteases (Naga and el-Shazly, 1968;Coleman, 1983;Forsberg et al., 1984;Nagasawa et al., 1994) and peptidases (Newbold et al., 1989), dietary proteins and peptides are more susceptible to cleavage, which will release free AA into the medium. Coleman (1975) proposed that RP utilize only about onehalf of their ingested N; the rest are expelled as shortchain peptides and free TAA. ...
... The HPLC determination of 2,6-diaminopimelic acid (1) and lanthionine (2) is complicated by the fact that these diamino acids occur as three stereoisomers (Fig. 1): a meso form (2R,6S), and a pair of enantiomers (2R,6R) and (2S,6S). Achiral chromatographic methods achieve a separation of the diastereomeric meso and rac forms Dugan et al., 1992;Mengin-Lecreulx et al., 1988;Puchaĺa et al., 1992;Richaud et al., 1987;Webster et al., 1990;Weir et al., 1989), whereas derivatization with a chiral reagent or use of a chiral solid support is needed to achieve separation of all three stereoisomers (El-Waziry et al., 1996;McKerrow et al., 2000;Nagasawa et al., 1993;Takahashi et al., 1989;Zanol and Gastaldo, 1991). Standard samples of the pure stereoisomeric forms of 1 and 2 are not readily available, making the assignment of the elution order of the diamino acid isomers difficult and limiting the interpretation of results obtained by chromatographic analysis. ...
... Chemical derivatization reagents such as dabsyl-chloride [43,87] and dansyl-chloride [88][89][90][91] are also applied in free amino acid bioanalytical assays, but less frequently than OPA and PITC. Fekkes discussed the application of other chemical derivatization reagents [27], such as fluorescamine [92][93][94][95] and phanquinone (4,7-phenanthroline-5,6-dione) [96,97]. Unfortunately, each of these chemical derivatization reagents had one or more analytical disadvantages compared with the standard chemical derivatization reagents . ...
... According to previous procedures, plasma 3-methyl-L-histidine concentration was measured with some modifications [13]. To 20 µL of plasma, 20 µL of internal standard (5 nmol/mL 1-Methyl-L-Histidine) and 40 µL of 20% trichloroacetic acid were added. ...