Structure-function analysis with tissue-type plasminogen activator. Effect of deletion of NH2-terminal domains on its biochemical and biological properties

Tissue-type plasminogen activator (t-PA) is a mosaic protein containing several distinct structural domains attached to the serine protease catalytic unit present at its COOH terminus. To investigate structure-function relationships in t-PA, we deleted the NH2-terminal domains, finger and epidermal growth factor, by genetic engineering. The genes for the parent and mutant t-PA were expressed in a bovine papilloma virus-dependent mammalian cell system. The secreted proteins were purified to homogeneity. The mutant protein was processed to the expected size of about 60 kDa compared to approximately 68 kDa for the parent t-PA, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fibrin autography. While the mutant t-PA had amidolytic activity comparable to native t-PA, it did not bind appreciably to fibrin. Consequently, fibrin-dependent enzymic activity, i.e. plasminogen activation in the presence of soluble fibrin and fibrinolysis were lower than with native recombinant t-PA. The effect of deletion of NH2-terminal domains on the plasma half-life (t1/2) was investigated by injecting native and mutant t-PA into mice. While the majority of the t-PA disappeared initially with a t1/2 of about 2 min, mutant t-PA cleared at a much slower rate with t1/2 of about 50 min. These findings suggest that the NH2-terminal domains of t-PA not only determine its specificity for binding to fibrin but also mediate its clearance from plasma in vivo. Furthermore, the catalytic unit in t-PA seems to function autonomously.     

An electronic mechanism in the action of drugs, ATP, transmitters and other cardinal adsorbents. II. Effect of ouabain on the relative affinities for Li+, Na+, K+, and Rb+ of surface anionic sites that mediate the entry of Cs+ into frog ovarian eggs

Ouabain enhanced the inhibitory effects of Li+, Na+, and K+ on the rate of Cs+ permeation into frog ovarian eggs while it reduced the inhibiting effect of Rb+. The data agree with earlier demonstrated effects of ouabain on the rank order of selective accumulation of the five alkali-metals in frog muscles and on the relative effectiveness of glycine, Li+, Na+, K+, Rb+, and Cs+ in inhibiting the rate of entry of Cs+ into frog sartorius muscle. In all three cases, the ouabain behaved as an electron-donating cardinal adsorbent (EDC) causing a rise of the electron density (c-value) of the beta- and gamma-carboxyl groups in the cell cytoplasm (for selective accumulation) and on the cell surface (for selective ion permeation). Explanations based on the association-induction hypothesis were offered why an EDC like ouabain does not initiate cell activation (like veratridine does) and why Ca++ and tetradotoxin delays or inhibits physiological and artificial cell activation.     

湖北贝母单鳞片砂培繁殖研究

本文针对湖北贝母生产中存在繁殖系数低的问题,研究了单鳞片砂培繁殖对提高鳞茎繁殖率的效果和原理。试验结果表明:1.单鳞片繁殖率为对照种鳞茎的5—9倍,2.低温(2—10℃)预处理4—8周和暗条件培养,能有效地提高子球形成率,促使子球迅速长大,3.植物激素(6-BA、KT、2,4-D)处理,有利于促进鳞片不定芽原基的分化,繁殖率为种茎繁殖的9—11倍;4.单鳞片繁殖的小鳞茎主要发生在鳞片基部的茎盘上,还可发生在鳞片的远轴面上,但不发生在近轴面。     

配体对(Na~++K~+)-ATP酶E_2→E_1转变的影响

 本研究确定了在0℃条件下,(Na~++K~+)-ATP酶纯化制备物与5mmol/L Na~+或Mg~(2+)在5mmol/L咪唑(pH7.4)环境中预保温30分钟,然后进行磷酸化,可以获得最高磷酸化水平,Na~+或Mg~(2+)的K_(0.5)值分别为0.29mmol/L或0.35mmol/L;以ADP代替Na~+和Mg~(2+)与酶预保温,对E_2向E_1转变无任何影响,而与Na~+、Mg~(2+)一起存在时则能加强Na~+及Mg~2的预保温效果。     

Control of nitrogenase inAzospirillum sp.

Many N₂-fixing organisms can turn off nitrogenase activity in the presence of NH₄ ⁺ and turn it on again when the NH₄ ⁺ is exhausted. One of the most interesting systems for accomplishing this is by covalent modification of one subunit of dinitrogenase reductase by dinitrogenase reductase ADP-ribosyltransferase (DRAT). The system can be reactivated when NH₄ ⁺ is exhausted, by dinitrogenase reductase activating glycohydrolase (DRAG) which removes the inactivating group. It is fascinating that some species of the genusAzospirillum possess the DRAT and DRAG systems (A. lipoferum andA. brasilense), whereasA. amazonense in the same genus lacks DRAT and DRAG.A. amazonense responds to NH₄ ⁺ but does not exhibit modification of dinitrogenase reductase characteristic of the action of DRAT. However, it has been possible to clone DRAT and DRAG and to introduce them intoA. amazonense, whereupon they become functional in this organism. The DRAT and DRAG system does not appear to function inAcetobacter diazotrophicus, an organism isolated from sugar cane, that fixes N₂ at a pH as low as 3.0.A. diazotrophicus does show a rather sluggish response to NH₄ ⁺. A level of about 10 M NH₄ ⁺ is required to switch off the system. The response to NH₄ ⁺ is influenced by the dissolved oxygen concentration (DOC) as has been reported forAzospirillum sp. A DOC in equilibrium with 0.1 to 0.2 kPa O₂ seems optimal for the response inA. diazotrophicus.     

Competitive inhibition of an energy-dependent nickel transport system by divalent cations in Bradyrhizobium japonicum JH.

Both nickel-specific transport and nickel transport by a magnesium transporter have been described previously for a variety of nickel-utilizing bacteria. The derepression of hydrogenase activity in Bradyzhizobium japonicum JH and in a gene-directed mutant of strain JH (in an intracellular Ni metabolism locus), strain JHK7, was inhibited by MgSO4. For both strains, Ni2+ uptake was also markedly inhibited by Mg2+, and the Mg(2+)-mediated inhibition could be overcome by high levels of Ni2+ provided in the assay buffer. The results indicate that both B. japonicum strains transport Ni2+ via a high-affinity magnesium transport system. Dixon plots (1/V versus inhibitor) showed that the divalent cations Co2+, Mn2+, and Zn2+, like Mg2+, were competitive inhibitors of Ni2+ uptake. The KiS for nickel uptake inhibition by Mg2+, Co2+, Mn2+, and Zn2+ were 48, 22, 12, and 8 microM, respectively. Cu2+ strongly inhibited Ni2+ uptake, and molybdate inhibited it slightly. Respiratory inhibitors cyanide and azide, the uncoupler carbonyl cyanide m-chlorophenylhydrazone, the ATPase inhibitor N,N'-dicyclohexylcarbodiimide, and ionophores nigericin and valinomycin significantly inhibited short-term (5 min) Ni2+ uptake, showing that Ni2+ uptake in strain JH is energy dependent. Most of these conclusions are quite different from those reported previously for a different B. japonicum strain belonging to a different serogroup.     

青甘边区黑河流域森林植被带及其昆虫区系的组成

黑河发源于青海、甘肃交界的祁连山区,是甘肃河西走廊最大的内陆河水系之一,水源丰富,流量稳定,这不仅取决于祁连山区大气降水和冰川融水,更重要的是与上游森林植被密不可分。但是,由于森林害虫猖獗成灾,严重威胁着这一地域森林的生存。近几年我们在森林昆虫普查的基础上,对黑河流域森林昆虫     

Influence of amino acids on nitrogen fixation ability and growth of Azospirillum spp.

The utilization of amino acids for growth and their effects on nitrogen fixation differ greatly among the several strains of each species of Azospirillum spp. that were examined. A. brasiliense grew poorly or not at all on glutamate, aspartate, serine, or histidine as the sole nitrogen and carbon sources. Nitrogen fixation by most A. brasiliense strains was inhibited only slightly even by 10 mM concentrations of these amino acids. In contrast, A. lipoferum and A. amazonense grew very well on glutamate, aspartate, serine, or histidine as the sole nitrogen and carbon sources; nitrogen fixation, which was measured in the presence of malate or sucrose, was severely inhibited by these amino acids. It was concluded that growth on histidine as the sole source of nitrogen, carbon, and energy may be used for the taxonomic characterization of Azospirillum spp. and for the selective isolation of A. lipoferum. The different utilization of various amino acids by Azospirillum spp. may be important for their establishment in the rhizosphere and for their associative nitrogen fixation with plants. The physiological basis for the different utilization of glutamate by Azospirillum spp. was investigated further. A. brasiliense and A. lipoferum exhibited a high affinity for glutamate uptake (Km values for uptake were 8 and 40 microM, respectively); the Vmax was 6 times higher in A. lipoferum than in A. brasiliense. At high substrate concentrations (10 mM), the nonsaturable component of glutamate uptake was most active in A. lipoferum and A. amazonense.(ABSTRACT TRUNCATED AT 250 WORDS)