Production of N‐Acetyl‐Phosphinothricin: A Substance used for Inducing Male Sterility in Transgenic Plants

The non‐toxic compound N‐acetyl‐L‐phosphinothricin (N‐Ac‐L‐PPT) is used in a so‐called deacetylation system to induce male sterility in transgenic plants by tapetum specific deacetylation to the herbicide L‐phosphinothricin (L‐PPT). A procedure was developed to produce pure racemic and L‐isomeric N‐Ac‐PPT containing less than 30 ppm residual PPT. Experiments applied to wild type tobacco and PPT‐resistant tobacco showed that the maximal tolerated N‐Ac‐PPT concentration would be less than 45 mM of the L‐isomer. Otherwise unspecific deacetylation by several acylases, as well as by environmental conditions like higher temperatures or pHs beyond neutrality, increased the residual L‐PPT content to toxic concentrations. In contrast, N‐acetyl‐L‐phosphinothricyl‐alanyl‐alanine (N‐Ac‐L‐PPTT), a substance also occurring during the biosynthesis of phosphinothricyl‐alanyl‐alanine (PPTT) by some Streptomyces species, was tolerated up to 274 mM by wild type tobacco plants. However, the ArgE deacatylase from Escherichia coli originally used in the deacetylation system, as well as some other acylases, showed no activity towards N‐Ac‐L‐PPTT.     

Structural comparison of the 68 kDa laminin-binding protein and 5'-nucleotidase from chicken muscular sources: evidence against a gross structural similarity of both proteins

The 68 kDa laminin-binding protein purified from chicken skeletal muscle and the ectoenzyme 5'-nucleotidase from chicken gizzard are both able to interact with laminin. They were both shown to possess a nearly identical amino acid composition. The 79 kDa glycosylated form of 5'-nucleotidase can be transformed into an enzymatically active form by treatment with endoglycosidase F (Endo F). Deglycosylated (Endo F-treated) 5'-nucleotidase exhibits an apparent molecular mass of 68 kDa. Using immunological and finger-printing techniques, both proteins were analysed to determine their structural relatedness. The results obtained indicate that both proteins are not identical but may posses a few common peptides of yet unknown sequence and length.     

Stability and reconstitution of pyruvate oxidase from Lactobacillus plantarum: dissection of the stabilizing effects of coenzyme binding and subunit interaction.

Pyruvate oxidase from Lactobacillus plantarum is a homotetrameric flavoprotein with strong binding sites for FAD, TPP, and a divalent cation. Treatment with acid ammonium sulfate in the presence of 1.5 M KBr leads to the release of the cofactors, yielding the stable apoenzyme. In the present study, the effects of FAD, TPP, and Mn2+ on the structural properties of the apoenzyme and the reconstitution of the active holoenzyme from its constituents have been investigated. As shown by circular dichroism and fluorescence emission, as well as by Nile red binding, the secondary and tertiary structures of the apoenzyme and the holoenzyme do not exhibit marked differences. The quaternary structure is stabilized significantly in the presence of the cofactors. Size-exclusion high-performance liquid chromatography and analytical ultracentrifugation demonstrate that the holoenzyme retains its tetrameric state down to 20 micrograms/mL, whereas the apoenzyme shows stepwise tetramer-dimer-monomer dissociation, with the monomer as the major component, at a protein concentration of < 20 micrograms/mL. In the presence of divalent cations, the coenzymes FAD and TPP bind to the apoenzyme, forming the inactive binary FAD or TPP complexes. Both FAD and TPP affect the quaternary structure by shifting the equilibrium of association toward the dimer or tetramer. High FAD concentrations exert significant stabilization against urea and heat denaturation, whereas excess TPP has no effect. Reconstitution of the holoenzyme from its components yields full reactivation. The kinetic analysis reveals a compulsory sequential mechanism of cofactor binding and quaternary structure formation, with TPP binding as the first step. The binary TPP complex (in the presence of 1 mM Mn2+/TPP) is characterized by a dimer-tetramer equilibrium transition with an association constant of Ka = 2 x 10(7) M-1. The apoenzyme TPP complex dimer associates with the tetrameric holoenzyme in the presence of 10 microM FAD. This association step obeys second-order kinetics with an association rate constant k = 7.4 x 10(3) M-1 s-1 at 20 degrees C. FAD binding to the tetrameric binary TPP complex is too fast to be resolved by manual mixing.     

Characterization of the stabilizing effect of point mutations of pyruvate oxidase from Lactobacillus plantarum: protection of the native state by modulating coenzyme binding and subunit interaction.

Point mutations in the gene of pyruvate oxidase from Lactobacillus plantarum, with proline residue 178 changed to serine, serine 188 to asparagine, and alanine 458 to valine, as well as a combination of the three single point mutations, lead to a significant functional stabilization of the protein. The enzyme is a tetrameric flavoprotein with tightly bound cofactors, FAD, TPP, and divalent metal ions. Thus, stabilization may be achieved either at the level of tertiary or quaternary interactions, or by enhanced cofactor binding. In order to discriminate between these alternatives, unfolding, dissociation, and cofactor binding of the mutant proteins were analyzed. The point mutations do not affect the secondary and tertiary structure, as determined by circular dichroism and protein fluorescence. Similarly, the amino acid substitutions neither modulate the enzymatic properties of the mutant proteins nor do they stabilize the structural stability of the apoenzymes. This holds true for both the local and the global structure with unfolding transitions around 2.5 M and 5 M urea, respectively. On the other hand, deactivation of the holoenzyme (by urea or temperature) is significantly decreased. The most important stabilizing effect is caused by the Ala-Val exchange in the C-terminal domain of the molecule. Its contribution is close to the value observed for the triple mutant, which exhibits maximum stability, with a shift in the thermal transition of ca. 10 degrees C. The effects of the point mutations on FAD binding and subunit association are interconnected. Because FAD binding is linked to oligomerization, the stability of the mutant apoenzyme-FAD complexes is increased. Accordingly, mutants with maximum apparent FAD binding exhibit maximum stability. Analysis of the quaternary structure of the mutant enzymes in the absence and in the presence of coenzymes gives clear evidence that both improved ligand binding and subunit interactions contribute to the observed thermal stabilization.     

Comparative effects of trichothecene mycotoxins on bovine platelet function: Acetyl T-2 toxin,a more potent inhibitor than T-2 toxin

The effects of the trichothecene mycotoxins (acetyl T-2 toxin, T-2 toxin, HT-2 toxin, palmityl T-2 toxin, diacetoxyscirpenol (DAS), deoxynivalenol (DON), and T-2 tetraol) on bovine platelet function were examined in homologous plasma stimulated with platelet activating factor (PAF). The mycotoxins inhibited platelet function with the following order of potency: acetyl T-2 toxin > palmityl T-2 toxin = DAS > HT-2 toxin = T-2 toxin. While T-2 tetraol was completely ineffective as an inhibitor, DON exhibited minimal inhibitory activity at concentrations above 10×10^?4M. The stability of the platelet aggregates formed was significantly reduced in all mycotoxin treated platelets compared to that of the untreated PAF controls. It is suggested that the increased sensitivity of PAF stimulated bovine platelets to the more lipophilic mycotoxins may be related to their more efficient partitioning into the platelet membrane compared to the more hydrophilic compounds.     

Drosophila pupal macrophages – A versatile tool for combined ex vivo and in vivo imaging of actin dynamics at high resolution

Molecular understanding of actin dynamics requires a genetically traceable model system that allows live cell imaging together with high-resolution microscopy techniques. Here, we used Drosophila pupal macrophages that combine many advantages of cultured cells with a genetic in vivo model system. Using structured illumination microscopy together with advanced spinning disk confocal microscopy we show that these cells provide a powerful system for single gene analysis. It allows forward genetic screens to characterize the regulatory network controlling cell shape and directed cell migration in a physiological context. We knocked down components regulating lamellipodia formation, including WAVE, single subunits of Arp2/3 complex and CPA, one of the two capping protein subunits and demonstrate the advantages of this model system by imaging mutant macrophages ex vivo as well as in vivo upon laser-induced wounding.     

吐鲁番市胃食管反流病发生相关危险因素分析（附280例报道）

目的：探讨280例胃食管反流病（GERD）的分布特点及危险因素。方法：对临床诊断和胃镜确诊的280例GERD患者进行临床和风险因子相关性分析。结果：不论汉族还是维族,男性患者比例均明显高于女性;汉族患者高发年龄段早于维族患者（z=-2．939,P=0．003,）;汉族和维族患者占反流性食管炎和Barrett食管比例分别为42．4％、81_3％及56．5％、18．8％,其中汉族患者Barrett食管比例较高（X2=14．358,P=0．000）;肥胖、习惯性便秘、重体力活动者、饮食习惯不良在维族患者中的比例较高（P〈0．001）。结论：GERD与性别、年龄密切相关,男性多于女性,汉族患者发病年龄高峰旱于维族患者;汉族患者Barrett食管发生比例高于维族患者;肥胖、习惯性便秘、重体力活动、饮食习惯不良可能是GERD尤其是维族人群GERD的危险因素。     

GAP promoter‐based fed‐batch production of highly bioactive core streptavidin by Pichia pastoris

Streptavidin is a homotetrameric protein binding the vitamin biotin and peptide analogues with an extremely high affinity, which leads to a large variety of applications. The biotin‐auxotrophic yeast Pichia pastoris has recently been identified as a suitable host for the expression of the streptavidin gene, allowing both high product concentrations and productivities. However, so far only methanol‐based expression systems have been applied, bringing about increased oxygen demand, strong heat evolution and high requirements for process safety, causing increased cost. Moreover, common methanol‐based processes lead to large proportions of biotin‐blocked binding sites of streptavidin due to biotin‐supplemented media. Targeting these problems, this paper provides strategies for the methanol‐free production of highly bioactive core streptavidin by P. pastoris under control of the constitutive GAP promoter. Complex were superior to synthetic production media regarding the proportion of biotin‐blocked streptavidin. The optimized, easily scalable fed‐batch process led to a tetrameric product concentration of up to 4.16 ± 0.11 µM of biotin‐free streptavidin and a productivity of 57.8 nM h^?1 based on constant glucose feeding and a successive shift of temperature and pH throughout the cultivation, surpassing the concentration in un‐optimized conditions by a factor of 3.4. Parameter estimation indicates that the optimized conditions caused a strongly increased accumulation of product at diminishing specific growth rates (μ ≈ D < 0.01 h^?1), supporting the strategy of feeding. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:855–864, 2016