A mutant hemolysin with lower biological activity produced by a mutant Vibrio parahaemolyticus

Abstract A mutant toxin (m-TDH) of thermostable direct hemolysin (Vp-TDH) of Vibrio parahaemolyticus w was isolated from the culture of a strain of this organism mutagenized with N -methyl- N '-nitro- N -nitrosoguanidine. Although the m-TDH had a molecular structure similar to the native Vp-TDH, the m-TDH retained only about 7% residual hemolytic activity of the native toxin. Furthermore, other biological activities of m-TDH, such as lethality in mice and enterotoxicity in rabbit ileal loops, were also weakened. The m-TDH was immunologically indistinguishable from the native Vp-TDH. These results suggest that the m-TDH is only slightly different in structure from the native Vp-TDH. Also, the mutagenized site in m-TDH, which is not immunogenic, seems to be involved in expressing not only hemolytic activity but also lethal and enterotoxic activity.     

Thrombin hydrolysis of an N-terminal peptide from fibrinogen Lille: kinetic and NMR studies.

Fibrinogen Lille, a congenital dysfibrinogenemia, has been reported to arise from a mutation from Asp to Asn at position 7 of the A alpha chain of human fibrinogen, thereby reducing the thrombin-catalyzed rate of hydrolysis of the Arg(16)-Gly(17) peptide bond of this chain. Synthetic peptides of relevant portions of the wild-type and mutant A alpha chains were prepared, and the thrombin-catalyzed rates of hydrolysis of their Arg(16)-Gly(17) peptide bonds were determined. In addition, transferred NOE measurements were made to deduce their conformations, when complexed to bovine thrombin. The kinetics data showed little difference in the hydrolysis rates between the wild-type and mutant peptides, and the NMR data indicate no difference in the bound conformation of these two peptides. Therefore, electrostatic (or salt-bridge) interactions between Asp(7) and thrombin do not influence the bound conformations of these peptides. Asp(7) may interact with a remote residue of fibrinogen, not present in these synthetic peptides, or there may be additional mutations beyond A alpha (1-20) which have not been detected in fibrinogen Lille. Alternatively, when thrombin binds to fibrinogen at its secondary binding site, its primary (active) site may display different reactivities toward wild-type fibrinogen and fibrinogen Lille.     

Conformational stability of a thrombin-binding peptide derived from the hirudin C-terminus.

The COOH-terminal region of hirudin represents an independent functional domain that binds to an anion-binding exosite of thrombin and inhibits the interaction of thrombin with fibrinogen and regulatory proteins in blood coagulation. The thrombin-bound structure of the peptide fragment, hirudin 55-65, has been determined by use of transferred NOE spectroscopy [Ni, F., Konishi, Y., & Scheraga, H. A. (1990) Biochemistry 29, 4479-4489]. The stability of the thrombin-bound conformation has been characterized further by a combined NMR and theoretical analysis of the conformational ensemble accessible by the hirudin peptide. Medium- and long-range NOE's were found for the free hirudin peptide in aqueous solution and in a mixture of dimethyl sulfoxide and water at both ambient (25 degrees C) and low (0 degrees C) temperatures, suggesting that ordered conformations are highly populated in solution. The global folding of these conformations is similar to that in the thrombin-bound state, as indicated by NOE's involving the side-chain protons of residues Phe(56), Ile(59), Pro(60), Tyr(63), and Leu(64). Residues Glu(61), Glu(62), Tyr(63), and Leu(64) all contain approximately 50% of helical conformations calculated from the ratio of the sequential dNN and d alpha N NOE's. Among the helical ensemble, active 3(10)-helical conformations were found by an analysis of the medium-range [(i,i+2) and (i,i+3)] NOE's involving the last six residues of the peptide. An analysis of the side-chain rotamers revealed that, upon binding to thrombin, there may be a rotation around the alpha CH-beta CH bond of Ile(59) such that Ile(59) adopts a gauche- (chi 1 = +60) conformation in contrast to the highly populated trans (chi 1 = -60) found for Ile(59) in the free peptide. However, the thrombin-bound conformation of the hirudin peptide is still an intrinsically stable conformer, and the preferred conformational ensemble of the peptide contains a large population of the active conformation. The apparent preference for a gauche- (chi 1 = +60) side-chain conformation of Ile(59) in the bound state may be explained by the existence of a positively charged arginine residue among the hydrophobic residues in the thrombin exosite.     

Two temporally synthesized charge subunits interact to form the five isoforms of cottonseed (Gossypium hirsutum) catalase.

Five charge isoforms of tetrameric catalase were isolated from cotyledons of germinated cotton (Gossypium hirsutum L.) seedlings. Denaturing isoelectric focusing of the individual isoforms in polyacrylamide gels indicated that isoforms A (most anodic) and E (most cathodic) consisted of one subunit of different charge, whereas isoforms B, C and D each consisted of a mixture of these two subunits. Thus the five isoforms apparently were formed through combinations of two subunits in different ratios. Labelling cotyledons in vivo with [35S]methionine at three daily intervals in the dark, and translation in vivo of polyadenylated RNA isolated from cotyledons at the same ages, revealed synthesis of two different subunits. One of the subunits was synthesized in cotyledons at all ages studied (days 1-3), whereas the other subunit was detected only at days 2 and 3. This differential expression of two catalase subunits helped explain previous results from this laboratory showing that the two anodic forms (A and B) found in maturing seeds were supplemented with three cathodic forms (C-E) after the seeds germinated. These subunit data also helped clarify our new findings that proteins of isoforms A, B and C (most active isoforms) accumulated in cotyledons of plants kept in the dark for 3 days, then gradually disappeared during the next several days, whereas isoforms D and E (least active isoforms) remained in the cells. This shift in isoform pattern occurred whether seedlings were kept in the dark or exposed to continuous light after day 3, although exposure to light enhanced this process. These sequential molecular events were responsible for the characteristic developmental changes (rise and fall) in total catalase activity. We believe that the isoform changeover is physiologically related to the changeover in glyoxysome to leaf-type-peroxisome metabolism.     

白百利烟草愈伤组织细胞细胞分裂素结合蛋白的研究

利用BA-Sepharose 4B亲和层析技术丛白百利烟草(Nicotiana tabacum Baibaili)愈伤组织细胞分离提纯了分子量为4400±100道尔顿的细胞分裂素结合蛋白(CB-蛋白)。在细胞表面、核糖体、线粒体、叶绿体和细胞核上以及在细胞液中都有CB-蛋白存在,而核糖体上的CB-蛋白含量量高。探讨了CB-蛋白的功能。     

家兔脊髓蛛网膜下腔注射乙酰胆碱对血压和心率的影响

将乙酰胆碱(ACh)注入麻醉家兔脊髓蛛网膜下腔,观察其对心血管活动的影响。结果表明:(1)脊髓蛛网膜下腔注射50～100μg ACh可使血压下降,心率减慢;(2)预先由脊髓蛛网膜下腔注射阿托品,可阻断ACh引起的降压和降心率作用;(3)脊髓蛛网膜下腔注射六甲双铵、酚妥拉明或心得安均不能阻断上述ACh的心血管反应;(4)切断两侧颈部迷走神经,ACh不再使心率减慢,但其降低血压的作用不受到任何影响。 脊髓中ACh水平升高可通过激活胆碱能M-受体引起血压下降和心率减慢。ACh的这种降压作用既没有中枢肾上腺素能受体活动参与,也不是通过迷走神经实现的,可能是由于脊髓交感血管中枢紧张性降低所造成的。     

Effects of social conditions on Juvenile Hormone mediated reproductive development in Bombus terrestris workers

Abstract. During the annual life cycle of the bumble bee Bombus terrestris (L.) colony, there is a stage characterized by worker reproduction in the presence of the queen. It has been proposed that this is a result of a decrease in queen inhibition. This hypothesis was examined by studying the effects of queens taken from colonies at different stages of development on several aspects of worker physiology and behaviour: rates of Juvenile Hormone (JH) release in vitro , ovary development, and behaviour associated with reproduction. After optimizing and validating the radiochemical assay for JH release for bumble bee workers, we found that queenless workers had significantly more developed ovaries and higher rates of release of JH than did queenright workers, confirming and extending previous findings that suggest that bumblebee ovarian development is under JH control. Mated queens, separated from their colony and brood, can have the same inhibitory effect on the reproductive development of callow workers. In contrast, workers confined with virgin queens or in queenless groups demonstrated a significantly higher rate of release of JH, overt aggression and threatening behaviours. However, there were no differences in rates of release of JH between workers confined in groups in the laboratory with queens taken from colonies either before or after the onset of worker reproduction. Furthermore, overt aggression and threatening behaviours were similar and low in both types of groups. These results gave no support to the hypothesis that a decrease in queen inhibition is associated with the onset of worker reproduction. We also show that young workers reared in colonies either before or after worker reproduction occurs, or in queenless colonies, all demonstrated similar, low rates of release of JH. These results suggest that older workers may inhibit the corpora allata of younger workers in queenless colonies.     

电刺激大鼠外侧缰核对延髓甩尾相关细胞的活动和甩尾反射的影响

在浅麻醉大鼠上,在延髓腹内侧结构内观察到三种具有不同放电类型的细胞,即乃尾前放电骤停的撤反应细胞,甩尾前放电骤增的给反应细胞和甩尾无关的中性细胞。电刺激外侧缰核可抑制撤反应细胞的自发放电,加强给反应细胞自发放电,从而易化两类细胞的甩尾相关反应,同时易化伤害刺激引起的甩尾反射。实验结果说明,外侧缰核对节段性防御反射有易化作用,这种易化作用可能是通过延髓内撤反应和反应细胞的协同活动而实现的。     

闽北地区四代三化螟发生动态Fuzzy预测模型研究

根据Fuzzy数学原理和三化螟生物学特性,组建了四代三化螟发生动态综合预测模型经对福建省将乐县16年四代三化螟发生期、发生量回报及1995年的预报,拟合率及正确率均达100％。     

多胚水稻ApⅢ（双13）的胚胎学观察

对多胚水稻（Ｏｒｙｚａ ｓａｔｉｖａ Ｌ．）ＡｐⅢ的大量成熟颖果、人工萌发的幼苗和开花后３～５ ｄ 的幼嫩颖果进行的整体解剖和显微制片观察表明：ＡｐⅢ的５０００粒成熟颖果中,８９．０％ 含单胚单苗,８．９％ 和１．２％分别含双胚双苗和三胚三苗;７００多粒幼嫩颖果中,９０．０％ ～９５．０％ 含单胚,５．０％ ～７．０％ 含双胚。因制片的数目有限,未见到含三胚的;在含单胚和多胚颖果中,胚均位于同一胚囊的珠孔端,未见到胚囊以外存在不定胚。根据上述结果,似可以认为ＡｐⅢ单粒颖果的双胚和三胚是由同一胚囊内的卵细胞和１或２个助细胞受精或不受精发育而来的