Mechanisms of caspase activation and inhibition during apoptosis

Apoptosis is primarily executed by active caspases, which are derived from the inactive procaspase zymogens through proteolytic cleavage. We determined the crystal structures of a caspase zymogen, procaspase-7, and an active caspase-7 without any bound inhibitors. Compared to the inhibitor-bound caspase-7, procaspase-7 zymogen exhibits significant structural differences surrounding the catalytic cleft, which precludes the formation of a productive conformation. Proteolytic cleavage in between the large and small subunits allows rearrangement of essential loops in the active site, priming active caspase-7 for inhibitor/substrate binding. Strikingly, binding by inhibitors causes a 180-degree-flipping of the N-terminus in the small subunit, which interacts with and stabilizes the catalytic cleft. These analyses reveal the structural mechanisms of caspase activation and demonstrate that the inhibitor/substrate binding is a process of     

Four residues of propeptide are essential for precursor folding of nattokinase

Subtilisin propeptide functions as an intramolecular chaperone that guides precursor folding. Nattokinase, a member of subtilisin family, is synthesized as a precursor consisting of a signal peptide, a propeptide, and a subtilisin domain, and the mechanism of its folding remains to be understood. In this study, the essential residues of nattoki- nase propeptide which contribute to precursor folding were determined. Deletion analysis showed that the con- served regions in propeptide were important for precursor folding. Single-site and multi-site mutagenesis studies con- firmed the role of Tyr10, Gly13, Gly34, and Gly35. During stage （i） and （ii） of precursor folding, Tyr10 and Gly13 would form the part of interface with subfilisin domain. While Gly34 and Gly35 connected with an α-helix that would stabil- ize the structure of propeptide. The quadruple Ala mutation, Y10A/G13A/G34A/G35A, resulted in a loss of the chaperone function for the propeptide. This work showed the essential residues of propeptide for precursor folding via secondary structure and kinetic parameter analyses.     

Improved method to raise polyclonal antibody using enhanced green fluorescent protein transgenic mice

Recombinant fusion protein is widely used as an antigen to raise antibodies against the epitope of a target protein. However, the concomitant anticarrier antibody in resulting antiserum reduces the production of the desired antibody and brings about unwanted non-specific immune reactions. It is proposed that the carrier protein transgeulc animal could be used to solve this problem. To validate this hypothesis, enhanced green fluorescent protein （EGFP） transgenic mice were produced. By immunizing the mice with fusion protein His6HAtag-EGFP, we showed that the antiserum from the transgenic mice had higher titer antibody against His6HA tag and lower titer antibody against EGFP compared with that from wild-type mice. Therefore, this report describes an improved method to raise high titer antipeptide polyclonal antibody using EGFP transgenic mice that could have application potential in antibody preparation.     

Analysis of essential histidine residues of maize branching enzymes by chemical modification and site-directed mutagenesis

Incubation of maize branching enzyme, mBEI and mBEII, with 100 μM diethylpyrocarbonate (DEPC) rapidly inactivated the enzymes. Treatment of the DEPC-inactivated enzymes with 100–500 mM hydroxylamine restored the enzyme activities. Spectroscopic data indicated that the inactivation of BE with DEPC was the result of histidine modification. The addition of the substrate amylose or amylopectin retarded the enzyme inactivation by DEPC, suggesting that the histidine residues are important for substrate binding. In maize BEII, conserved histidine residues are in catalytic regions 1 (His320) and 4 (His508). His320 and His508 were individually replaced by Ala via site-directed mutagenesis to probe their role in catalysis. Expression of these mutants inE. coli showed a significant decrease of the activity and the mutant enzymes hadK _m values 10 times higher than the wild type. Therefore, residues His320 and His508 do play an important role in substrate binding.     

Negative regulation of caspase 3-cleaved PAK2 activity by protein phosphatase 1

The p21-activated kinase 2 (PAK2) is activated by binding of small G proteins, Cdc42 and Rac, or through proteolytic cleavage by caspases or caspase-like proteases. Activation by both small G protein and caspase requires autophosphorylation at Thr-402 of PAK2. Although activation of PAK2 has been investigated for nearly a decade, the mechanism of PAK2 downregulation is unclear. In this study, we have applied the kinetic theory of substrate reaction during modification of enzyme activity to study the regulation mechanism of PAK2 activity by the catalytic subunit of protein phosphatase 1 (PP1α). On the basis of the kinetic equation of the substrate reaction during the reversible phosphorylation of PAK2, all microscopic kinetic constants for the free enzyme and enzyme-substrate(s) complexes have been determined. The results indicate that (1) PP1α can act directly on phosphorylated Thr-402 in the acti-vation loop of PAK2 and down-regulate its kinase activity; (2) binding of the exogenous protein/peptide substrates at the active site of PAK2 decreases both the rates of PAK2 autoactivation and inactivation. The present method provides a novel approach for studying reversible phosphorylation reactions. The advantage of this method is not only its usefulness in study of substrate effects on enzyme modifica-tion but also its convenience in study of modification reaction directly involved in regulation of enzyme activity. This initial study should provide a foundation for future structural and mechanistic work of protein kinases and phosphatases.     

浑善达克沙地典型灌木和草本植物对模拟降雨的不同反应(英文)

To assess the ecophysiological responses and adaptive strategies to rainfall exhibited by different plant functional types, we compared gas exchange, leaf water potential (ψleaf) and PS photochemical efficiency (Fv/Fm) between Hedysarum fruticosum Pall. (shrub) and Salsola collina Y. L. Chang(herb) under a series of simulated rainfalls. The experiment was conducted in Hunshandak Sandy Land, North China. The values of net photosynthetic rate (Pn), transpiration rate (E), stomatal conductance (gs), Fv/Fm and ψleaf always elevated with the increasing amount of simulated rainfall. Whereas water useefficiency (WUE) maintained constant in H. fruticosum while always decreased in S.collina. Both speciesfully recovered their Pn and Fv/Fm 3d after rainfall, while gs and ψleaf recovered only within 2 d. However, the response patterns were obviously different. H.fruticosu promote its physiological traits within 5-15 mm rainfall but no further positive enhancement was noted when rainfall was larger than 15 mm. In S.collina, however, the enhancement continued with more water applied. We inferred that H.fruticosum and S.collina responded similarly to rainfall in time courses but differential to simulated precipitation change. S.collina was likely to be physiologically sensitive to instant increasing soil water, that can be regarded as a prodigal water use type; whilst H.fruticosum was a conservative water user.     

Urokinase mutant with better fibrin-specificity

A 150 -156 amino acids-deleted single-chain urokinase-type plasminogen activator (dscu-PA) and its rccombinant wild-type counterpart (rscu-PA) were both expressed in Escherichia coli. After denaturation and renaturation in nitro, the expressed products were both purified lo a single silver-stained band by means of IgG affinity chromatography. After activation by plasrran, similar enzymatic constants based on the hydrolysis of synthetic substrate S2444 by the two-chain molecular forms of dscu-PA and rscu-PA, or native tcu-PA were observed, suggesting that no impairment had been exerted on the catalytic active site of dtcu-PA by the 150-156 amino acids deletion. In both in vitro fibrin-clot and 125I-fibrin sepharose lysis tests, dtcu-PA showed a significantly higher fibrinolytic activity than rtcu-PA or rscu-PA. Hardly any effect on the concentration of fibrinogen in plasma was found in dtcu-PA. It was concluded that dtcu-PA had a higher fibrin specificity and that tcu-PA could be provided with better fib     

Reducing Viral Inhibition of Host Cellular Apoptosis Strengthens the Immunogenicity and Protective Efficacy of an Attenuated HSV-1 Strain

Herpes simplex virus 1(HSV-1), a member of a herpesviruses, shows a high infectivity rate of 30%–60% in populations of various ages. Some herpes simplex(HSV) vaccine candidates evaluated during the past 20 years have not shown protective efficacy against viral infection. An improved understanding of the immune profile of infected individuals and the associated mechanism is needed. HSV uses an immune evasion strategy during viral replication, and various virus-encoded proteins, such as ICP47 and Vhs, participate in this process through limiting the ability of CD8?cytotoxic T lymphocytes to recognize target cells. Other proteins, e.g., Us3 and Us5, also play a role in viral immune evasion via interfering with cellular apoptosis. In this work, to study the mechanism by which HSV-1 strain attenuation interferes with the viral immune evasion strategy, we constructed a mutant strain, M5, with deletions in the Us3 and Us5 genes. M5 was shown to induce higher neutralizing antibody titers and a stronger cellular immune response than our previously reported M3 strain,and to prevent virus infection more effectively than the M3 strain in an in vivo mouse challenge test.     

Enlarged fins of Tibetan catfish provide new evidence of adaptation to high plateau

The uplift of the Tibetan Plateau significantly altered the geomorphology and climate of the Euroasia by creating large mountains and rivers. Fishes are more likely to be affected relative to other organisms, as they are largely restricted to river systems. Faced with the rapidly flowing water in the Tibetan Plateau, a group of catfish has evolved greatly enlarged pectoral fins with more numbers of fin-rays to form an adhesive apparatus. However, the genetic basis of these adaptations in Tibetan...     

Seasonal changes on two different spatial scales： response of aquatic invertebrates to water body and microhabitat

Knowledge about the spatial and temporal scales of both habitat use and the functional significance of different adaptations is essential for an understanding of the population dynamics of invertebrate assemblages. This fundamental knowledge is not only interesting from an academic point of view, but is sorely lacking and needed in the field of restoration ecology. Many species are threatened due to degradation. Knowing what environmental conditions are needed dtLring the life cycle of these species is important in the design of restoration measures which aim to lift existing bottlenecks for threatened species. To assess the relative importance of water type and microhabitat in structuring the invertebrate assemblage during different seasons, invertebrates were sampled in three water bodies differing in trophic level and acidity. Different parts within a water body （microhabitats） were sampled separately and each water body was sampled in all four seasons. Results show that water body is an important factor structuring the invertebrate assemblage early in the season, whereas microhabitat became more important later in the season. Structural complexity of microhabitats was related to the type of locomotion employed by invertebrates. Seasonal differences could be related to population dynamics （reproduction, mortality）. Moreover, fluctuations in resource availability were expected to differ between the water bodies, with highest fluctuations in the eutrophic water body and with fluctuations becoming less predictable later in the season. This was confirmed by the data： species synchronization to pulses in food availability was strongest in the eutrophic water body. Moreover, synchronization was strongest in summer, while in autumn waters were invaded by dispersive species. Based on these results a synthesis is presented on the functioning of the different waters during the different seasons.     

Drought-Stimulated Activity of Plasma Membrane Nicotinamide Adenine Dinucleotide Phosphate Oxidase and Its Catalytic Properties in Rice

The activity of plasma membrane （PM） nicotinamide adenine dinucleotide phosphate （NADPH） oxidase and its catalytic properties in rice was investigated under drought stress conditions. Drought stress led to decreased leaf relative water content （RWC） and, as a result of drought-induced oxidative stress, the activities of antioxidant enzymes increased significantly. More interestingly, the intensity of applied water stress was correlated with increased production of H2O2 and O2^- and elevated activity of PM NADPH oxidase, a key enzyme of reactive oxygen species generation in plants. Histochemical analyses also revealed increased H2O2 and O2^- production in drought-stressed leaves. Application of diphenylene iodonium （DPI）, an inhibitor of PM NADPH oxidase, did not alleviate drought-induced production of H2O2 and O2^-. Catalysis experiments indicated that the rice PM NADPH oxidase was partially fiavin-dependent. The pH and temperature optima for this enzyme were 9.8 and 40 ℃, respectively. In addition, drought stress enhanced the activity under alkaline pH and high temperature conditions. These results suggest that a complex regulatory mechanism, associated with the NADPH oxidase-H2O2 system, is involved in the response of rice to drought stress.     

Two-Dimensional Aerodynamic Models of Insect Flight for Robotic Flapping Wing Mechanisms of Maximum Efficiency

In the ＂modified quasi-steady＂ approach, two-dimensional （2D） aerodynamic models of flapping wing motions are analyzed with focus on different types of wing rotation and different positions of rotation axis to explain the force peak at the end of each half stroke. In this model, an additional velocity of the mid chord position due to rotation is superimposed on the translational relative velocity of air with respect to the wing. This modification produces augmented forces around the end of each stroke. For each case of the flapping wing motions with various combination of controlled translational and rotational velocities of the wing along inclined stroke planes with thin figure-of-eight trajectory, discussions focus on lift-drag evolution during one stroke cycle and efficiency of types of wing rotation. This ＂modified quasi-steady＂ approach provides a systematic analysis of various parameters and their effects on efficiency of flapping wing mechanism. Flapping mechanism with delayed rotation around quarter-chord axis is an efficient one and can be made simple by a passive rotation mechanism so that it can be useful for robotic application.     

Persistence of VRC01-resistant HIV-1 during antiretroviral therapy

VRC01,a broadly neutralizing monoclonal antibody(bnmAb),can neutralize a diverse array of HIV-1 isolates by mimicking CD4 binding to the envelope glycoprotein gp120.We have previously demonstrated the presence of VRC01-resistant strains in an HIV-1 infected patient during antiretroviral therapy.Here,we report follow-up studies of two subsequent samples from the same patient.With genetic and phenotypic analysis of over 70 full-length molecular clones of the HIV-1 envelope,we show that VRC01-resistant HIV-1 continued to exist and change in its proportion of the infecting virus during treatment with a highly active antiretroviral therapy.Consistent with our previous observation,the resistant phenotype was associated with a single asparagine residue at position 460(N460),a potential N-linked glycosylation site in the V5 region.The persistence and continuing evolution of VRC01-resistant HIV-1 in vivo presents a great challenge to our future preventative and therapeutic interventions based on VRC01.     

Deciphering the Stigmatic Transcriptional Landscape of Compatible and Self-Incompatible Pollinations in Brassica napus Reveals a Rapid Stigma Senescence Response Following Compatible Pollination

Dear Editor, Self-incompatibility （SI） is a genetic mechanism through which flowering plants prevent self-pollination to ensure out- crossing and genetic diversity. In Brassica sp., this mechanism is controlled by the self-incompatibility （S） locus, in which, the stigmatic ＇S-locus receptor kinase （SRK）＇ recognizes the ＇S-locus cysteine rich protein （SCR）＇ from the self-pollen to elicit an active rejection response. This results in blocking of compatibil- ity factors from being delivered to the site of pollen attachment leading to self-pollen rejection （Chapman and Goring, 2010）. In contrast, following recognition of compatible signals from the cross-pollen or compatible pollen （CP）, the stigma releases its resources such as water and nutrients to the dry pollen so that the pollen tube can germinate and penetrate the stigmatic cuticle leading to successful fertilization. Thus, an incompatible or self-pollen is fully capable of eliciting a compatible response, but is actively rejected before compatible responses can occur.     

Immunological dynamics in response to two anthrax vaccines in mice

In order to understand the variation of humoral and cellular immune responses to A16R live spore and AVA vaccine and to identify efficient immunological parameters for the early evaluation of post immu- nization in mice, we dynamically monitored the antibody production and cellular responses after the vaccination of Balb/C mice with the anthrax vaccines. The results show that both anti-AVA and anti-Spore antibodies were detectable in the A16R live spore vaccinated group while high titers of anti-AVA antibodies but not anti-Spore antibodies existed in the AVA-immunized group. IgG1 and IgG2 were the major subtypes of IgG in both of the two groups. However, the IgG2a level was significantly higher in the A16R group than in the AVA group. At the cellular level, responses of antigen-specific TH2, TH1 and plasma cells were detected. The peripheral TH2 responses could be seen on day 5 after vac- cination, and remained at a high level throughout the experiment (from day 5 post primary immuniza- tion to day 60 post the tertiary immunization); the TH1 responses to A16R vaccine appeared on day 5, while the responses to AVA could only be detected by day 7 after the secondary immunization; a low level of TH1 responses could be observed at the end of the experiment. Antigen-specific plasma cells could be found in the peripheral blood of both the immunized groups, however, the responses in the A16R group appeared earlier, lasted longer, and shown an ascending tendency until the end of the ex- periment when the plasma cell responses in the AVA group were reduced to a very low level. The re- sults suggest that the multiple antigen containing A16R live spore vaccine induces better immune re- sponses than AVA. Combined with serum antibody titers, TH2, TH1 and plasma cell responses could be used as immunological parameters for the evaluation of vaccine efficacy. These findings may afford new insight into the early evaluation of vaccination as well as being a powerful strategy for vaccine development.     

Protective mechanism of desiccation tolerance in Reaumuria soongorica: Leaf abscission and sucrose accumulation in the stem

Reaumuria soongorica (Pall.) Maxim., a perennial semi-shrub, is widely found in semi-arid areas in northwestern China and can survive severe desiccation of its vegetative organs. In order to study the protective mechanism of desiccation tolerance in R. soongorica, diurnal patterns of net photosynthetic rate (Pn), water use efficiency (WUE) and chlorophyll fluorescence parameters of Photosystem II (PSII), and sugar content in the source leaf and stem were investigated in 6-year-old plants during progressive soil drought imposed by the cessation of watering. The results showed that R. soongorica was char-acterized by very low leaf water potential, high WUE, photosynthesis and high accumulation of sucrose in the stem and leaf abscission under desiccation. The maximum Pn increased at first and then de-clined during drought, but intrinsic WUE increased remarkably in the morning with increasing drought stress. The maximal photochemical efficiency of PSII (Fv/Fm) and the quantum efficiency of noncyclic electric transport of PSII(ΦPSII) decreased significantly under water stress and exhibited an obvious phenomenon of photoinhibition at noon. Drought stressed plants maintained a higher capacity of dis-sipation of the excitation energy (measured as NPQ) with the increasing intensity of stress. Conditions of progressive drought promoted sucrose and starch accumulation in the stems but not in the leaves. However, when leaf water potential was less than –21.3 MPa, the plant leaves died and then abscised. But the stem photosynthesis remained and, afterward the plants entered the dormant state. Upon re-watering, the shoots reactivated and the plants developed new leaves. Therefore, R. soongorica has the ability to reduce water loss through leaf abscission and maintain the vigor of the stem cells to survive desiccation.     

三疣梭子蟹卵附着机制及相关形态学特征

Using histological methods and scanning electron microscopy, the structure of cement gland and formation of outer egg-membrane and egg-stalk of the swimming crab Portunus trituberculatus were studied to explain the mechanism of egg-attachment and function of the cement gland. Eggs adhered to the setae of pleopodal basipodite and endopodite for hatching followed by spawning but no egg attached on the setae of exopodite. The setae of pleopodal basipodite and endopodite were smooth for egg attachment while the setae of exopodite were branched and suitable for collecting and protecting eggs. Cement gland distributed along longitudinal axis of the pleopodal basipodite and endopodite under epithelia cells. Cells of the cement gland were elliptical and its size was about 50 times of the epithelium. Some glue secreted to the surface of the pleopod along the seta, while the other glue secreted to the surface of the pleopod by the channel which across the integument of the pleopod. The first spawned egg was transferred to the setae that were far from the pore, not attached to the setae of the pleopodal basipodite and endopodite near the genital pore. And the egg attachment became nearer and nearer to the pore with further spawning. Since the eggs to glide rather than roll, there was almost no glue on the contrary surface of the egg at first: and with further secretion, the whole surface was surrounded by glue. The thick part of glue formed one short bud during the glide of the egg and gradually developed into a full egg-stalk. On the other hand, the else glue developed into an outer egg-membrane, which was “trichromatic egg membrane” originally. Each egg attached to the setae of pleopodal basipodite and endopodite with its own egg-stalk. The egg-stalk was a plane strap at the formation stage and later it rolled into a rope shape, but it could be reverted to the former by outside force. Four types of egg attachment : an egg to a seta, an egg to several setae, several eggs to a seta and several eggs to several setae were discovered. In summary, the cement gland existed in the pleopod of the swimming crab and formed outer egg membrane and egg-stalk during the egg attachment [Acta Zoologica Sinica 50 (5) : 873 - 879, 2004].     

Structural-Nanomechanical Property Correlation of Shallow Water Shrimp （Pandalus platyceros） Exoskeleton at Elevated Temperature

This investigation reports the nanomechanical properties of shallow water shrimp exoskeleton at temperatures ranging from 30 ℃ to 80 ℃ measured using nanoindentation experiments. Scanning Electron Microscopy （SEM） measurements suggest that the shrimp exoskeleton has the Bouligand structure in its layers, a key characteristic of the crustaceans. The thickness of the layers and packing density are found to be different from that of lobsters and crabs reported earlier in the literature. Mechanical properties at high temperatures are determined using micro materials nanoindentation test set up combined with the hot stage. The properties measured during nanoindentation test are corrected for the creep and thermal drift during the experiments. The reduced modulus values are found to be around 28 GPa at 30 ℃ that reduces to approximately 24 GPa at 80 ℃. The hardness values also decrease from 1.6 GPa at 30 ℃ to around 1.2 GPa at 80 ℃. The indentation size effect is found to be absent at all temperatures. Creep mechanisms of polymers like materials and its temperature dependence are discussed to give more insight into the deformation mechanism.     

Crystal structure of Arabidopsis thaliana RabA_(1a)

RabGTPase is a member of the Ras superfamily of small GTPases, which share a GTP-binding pocket containing highly conserved motifs that promote GTP hydrolysis. In Arabidopsis, the RabA group, which corresponds to the Rab_(11) group in animals, functions in the recycling of endosomes that control docking and fusion during vesicle transport. However, their molecular mechanisms remain unknown. In this study, we determined the crystal structures of the GDP-bound inactive form and both GppNHp-and GTP-bound active forms of RabA1a, at resolutions of 2.8, 2.6, and 2.6 A?,respectively. A bound sulfate ion in the active site of the GDP-bound structure stabilized Switch Ⅱ by bridging the interaction between a magnesium ion and Arg74.Comparisons of the two states of RabA1a with Rab_(11) proteins revealed clear differences in the Switch Ⅰ and Ⅱ loops. These results suggested that conformational change of the Switch regions of RabA1a, derived by GTP or GDP binding, could maintain subcellular membrane traffic through the specific interaction of effector molecules.