Hideya Homma

Purification and Characterization of a Hemolysin-Like Protein, Sll1951, a Nontoxic Member of the RTX Protein Family from the Cyanobacterium Synechocystis sp. Strain PCC 6803

The hemolysin-like protein (HLP) Sll1951, characterized by the GGXGXDXUX nonapeptide motif implicated in Ca(2+) binding, was purified from the glucose-tolerant strain (GT) of Synechocystis sp. strain PCC 6803. HLP was eluted at 560 kDa after gel filtration chromatography. Atomic absorption spectroscopy indicated that the protein bound calcium. The bound Ca(2+) was not chelated with EGTA; however, it was released after being heated at 100 degrees C for 1 min, and it rebound to the Ca(2+)-depleted protein at room temperature. The apparent HLP molecular mass increased to 1,000 kDa and reverted to 560 kDa during the release and rebinding of Ca(2+), respectively. The monomers of the respective forms appeared at 90 and 200 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. HLP showed no apparent hemolytic activity against sheep erythrocytes; however, a slight hemolytic activity was detected during the conformational change caused by the rebinding of Ca(2+). Immunoelectron microscopy using polyclonal antibodies against the 200-kDa monomer revealed that HLP is located in the cell surface layer. The localization and Ca(2+)-induced reversible conformational change suggest that HLP is a member of the repeat in toxin (RTX) protein family despite its latent and low toxicity. In some other cyanobacteria, RTX proteins are reported to be necessary for cell motility. However, the GT was immotile. Moreover, the motile wild-type strain did not express any HLP, suggesting that HLP is one of the factors involved in the elimination of motility in the GT. We concluded that the involvement of RTX protein in cyanobacterial cell motility is not a general feature.

Ruminal Liquid Turnover Rate and Saliva Flow in Buffaloes and Holstein Cattle

イナワラ給与で,水牛のルーメン内液相回転率は,液相指標物質としてCo-HDTAを用いて測定すると牛よりも遅くなることが知られている.この種間差の生じる理由を明らかにするために実験を行なった.泌乳していない成雌水牛とホルスタイン牛の各3頭ににチモシー乾草の自由摂取量を1日8回に分けて3時間毎に給与した.Co-EDTAを経口投与後,3時間毎に8回ルーメン液を採取し,Co濃度の減少曲線からルーメン液相回転率を求め,更に,唾液流量(ルーメン液相流出量-水分摂取量)も求めた.この結果,イナワラ給与時と同様にチモシー乾草給与でも水牛のルーメン液相回転率は牛よりも遅い傾向が認められた.また,唾液流量は牛の半分の値であった.ルーメン液の無機りん酸塩,アンモニア態窒素および尿素態窒素の濃度はいずれも水牛の方が高かった.にれは水牛の唾液中のこれらの化学成分の濃度が隼よりも高いことを反映していると考えるにとができる.しかし,水牛のルーメン液の無機りん酸塩濃度は,唾液中の高濃度を考慮しても,唾液流量が牛の半分とすると,説明のつかない高すぎる値であった.この矛盾は,水牛のルーメン壁からの水分吸収率が牛よりも高いために,ルーメン内容物が濃縮されて生じると仮定すると説明できる.また,この板説を採用すると,水牛が牛よりもルーメン液相回転率が遅いことや,唾液流量が少ないことの理由は,指標物質が濃縮されたために過少に推定された見かけ上の結果であると解釈できる.更に,このルーメン内容物の濃縮は水牛のルーメン内で見られる細菌の高濃度や遅い飼料通過速度の原因であると考えられる.