A comprehensive test of the ‘limiting resources’ framework applied to plant tolerance to apical meristem damage

Tolerance to apical meristem damage (AMD) is a form of plant defense against herbivory. Theoretical models come to different conclusions about the effects of inorganic soil nutrient levels on tolerance to AMD, and different plants have shown different relationships between these variables. To assign some order to these disparate patterns and to resolve conflicts among the models, the ‘limiting resources model’ (LRM) was developed. However, we believe that the LRM is actually comprised of several different models, which we describe. Our study marks the first comprehensive and simultaneous test of the entire LRM framework, treating it explicitly as separate models, which also evaluates the models’ underlying assumptions. We studied tolerance to AMD in laboratory‐reared natural populations of Arabidopsis thaliana from three different regions of Europe, spanning a wide latitudinal gradient. We show that, in different populations of this species, basic responses to nutrients and damage are best described by different models, which are based on different assumptions and make different predictions. This demonstrates the need for complexity in our explanations, and suggests that no one existing model can account for all relationships between tolerance to AMD and nutrients. Our results also demonstrate that fruit production can provide a misleading approximation of fitness in A. thaliana, contrary to the common assumption in the literature.     

A plasmid display platform for the selection of peptides exhibiting a functional cell-penetrating phenotype

Cell-penetrating peptides (CPPs) represent a promising nonviral platform for the delivery of therapeutic cargos to cells and tissues. However, these peptides are often nonspecific, and their mechanism of action is still a subject of debate, which hinders the design of new CPPs. The alternative to rational protein design is the combinatorial approach to protein engineering, whereby large libraries of peptides are created and a screening or selection procedure is used to identify members with the desired phenotype(s). Here we describe a novel procedure for selecting peptides with a CPP phenotype using a plasmid display (PD) platform to link the peptides to their encoding DNA sequences. The PD system is based on genetic fusions to a DNA binding domain. The plasmid was designed to concomitantly express a fluorescent reporter protein to serve as a mock therapeutic cargo indicating its functional delivery into a cell. We have demonstrated this selection strategy using a control CPP (the TAT peptide) in the PC12 neuronal-like cell line. In the absence of transfection reagents, TAT was unable to deliver the protein/DNA complexes. The inclusion of the HA2 peptide from the hemagglutinin protein and the addition of polyethylenimine (PEI) were similarly ineffective. The addition of Lipofectamine, however, enabled the TAT-mediated delivery of the protein/DNA complexes, which was significantly better than control experiments without a CPP. This new PD selection platform will be a valuable new approach for use in identifying unique CPPs from randomized libraries with novel abilities and specificities.     

Germ band retraction as a landmark in glucose metabolism during <Emphasis Type="Italic">Aedes aegypti</Emphasis> embryogenesis

Background  

The mosquito A. aegypti is vector of dengue and other viruses. New methods of vector control are needed and can be achieved by a better understanding of the life cycle of this insect. Embryogenesis is a part of A. aegypty life cycle that is poorly understood. In insects in general and in mosquitoes in particular energetic metabolism is well studied during oogenesis, when the oocyte exhibits fast growth, accumulating carbohydrates, lipids and proteins that will meet the regulatory and metabolic needs of the developing embryo. On the other hand, events related with energetic metabolism during A. aegypti embryogenesis are unknown.     

大鼠心肌缺血再灌注损伤模型的实验研究

目的 制备一种新型的心肌急性缺血再灌注损伤模型,以探讨一种更符合临床实际需求的实验方法.方法 将20只雌性SD(Sprague-Dawley)大鼠随机分成2组(对照组、实验组),采用结扎主动脉根部引起心肌缺血5min再灌注30 min建立心肌急性缺血再灌注模型;通过应用透射电镜观察心肌细胞超微结构的改变,同时检测心肌组织匀浆丙二醛(Maleic Dialdehyde,MDA)含量、超氧化物歧化酶(Superoxide Dismutase,SOD)活力.结果 透射电镜下超微结构显示实验组较对照组明显加重了心肌组织结构和线粒体的损害;实验组心肌组织MDA明显高于对照组(P<0.01),而SOD明显低于对照组(P<0.01).结论 本实验成功建立了方法简便、易于操作、取材范围广泛的心肌缺血再灌注损伤模型,为心肌缺血再灌注损伤研究提供了一种更为可行的模型.     

Merriam's kangaroo rats (Dipodomys merriami) voluntarily select temperatures that conserve energy rather than water

Desert endotherms such as Merriam's kangaroo rat (Dipodomys merriami) use both behavioral and physiological means to conserve energy and water. The energy and water needs of kangaroo rats are affected by their thermal environment. Animals that choose temperatures within their thermoneutral zone (TNZ) minimize energy expenditure but may impair water balance because the ratio of water loss to water gain is high. At temperatures below the TNZ, water balance may be improved because animals generate more oxidative water and reduce evaporative water loss; however, they must also increase energy expenditure to maintain a normal body temperature. Hence, it is not possible for kangaroo rats to choose thermal environments that simultaneously minimize energy expenditure and increase water conservation. I used a thermal gradient to test whether water stress, energy stress, simultaneous water and energy stress, or no water/energy stress affected the thermal environment selected by D. merriami. During the night (i.e., active phase), animals in all four treatments chose temperatures near the bottom of their TNZ. During the day (i.e., inactive phase), animals in all four treatments settled at temperatures near the top of their TNZ. Thus, kangaroo rats chose thermal environments that minimized energy requirements, not water requirements. Because kangaroo rats have evolved high water use efficiency, energy conservation may be more important than water conservation to the fitness of extant kangaroo rats.     

Alteration of the specificity of the cofactor-binding pocket of Corynebacterium 2,5-diketo-D-gluconic acid reductase A

The NADPH-dependent 2,5-diketo-D-gluconic acid (2,5-DKG) reductase enzyme is a required component in some novel biosynthetic vitamin C production processes. This enzyme catalyzes the conversion of 2,5-DKG to 2-keto-L-gulonic acid, which is an immediate precursor to L-ascorbic acid. Forty unique site-directed mutations were made at five residues in the cofactor-binding pocket of 2,5-DKG reductase A in an attempt to improve its ability to use NADH as a cofactor. NADH is more stable, less expensive and more prevalent in the cell than is NADPH. To the best of our knowledge, this is the first focused attempt to alter the cofactor specificity of a member of the aldo-keto reductase superfamily by engineering improved activity with NADH into the enzyme. Activity of the mutants with NADH or NADPH was assayed using activity-stained native polyacrylamide gels. Eight of the mutants at three different sites were identified as having improved activity with NADH. These mutants were purified and subjected to a kinetic characterization with NADH as a cofactor. The best mutant obtained, R238H, produced an almost 7-fold improvement in catalysis with NADH compared with the wild-type enzyme. Surprisingly, most of this catalytic improvement appeared to be due to an improvement in the apparent kcat for the reaction rather than a large improvement in the affinity of the enzyme for NADH.     

Stability of the Agrobacterium tumefaciens VirB10 Protein Is Modulated by Growth Temperature and Periplasmic Osmoadaption

Export of oncogenic T-DNA from the phytopathogen Agrobacterium tumefaciens is mediated by the products of the virB operon. It has recently been reported (K. J. Fullner and E. W. Nester, J. Bacteriol. 178:1498–1504, 1996) that DNA transfer does not occur at elevated temperatures; these observations correlate well with much earlier studies on the temperature sensitivity of crown gall tumor development on plants. In testing the hypothesis that this loss of DNA movement reflects a defect in assembly or maintenance of a stable DNA transfer machinery at high temperature, we have found that steady-state levels of VirB10 are sensitive to growth temperature while levels of several other VirB proteins are considerably less affected. This temperature-dependent failure to accumulate VirB10 is exacerbated in an attachment-deficient mutant strain (chvB) which exhibits pleiotropic defects in periplasmic osmoadaption, and virulence of a chvB mutant can be partially restored by lowering the temperature at which the bacteria and the plant tissue are cocultivated. Furthermore, the stability of VirB10 is diminished in cells lacking functional VirB9, but only under conditions of low osmolarity. We propose that newly synthesized VirB10 is inherently labile in the presence of a large osmotic gradient across the inner membrane and is rapidly degraded unless it is stabilized by VirB9-dependent assembly into oligomeric complexes. The possibility that VirB10-containing complexes are not assembled properly at elevated temperatures suggests an explanation for the decades-old observation that tumor formation is exquisitely sensitive to ambient temperature.