A fungal enzyme with the ability of aflatoxin B₁ conversion: purification and ESI-MS/MS identification

Armillariella tabescens, a Chinese edible and medicinal fungus, whose multienzyme exist ability of AFB₁-converting, and ADTZ (aflatoxin-detoxizyme) had previously purified from the A. tabescens multienzyme monitored by AFB₁ conversion_. However, the enzyme now confirmed an oxidase and renamed aflatoxin-oxidase (AFO). In this paper, AFO was purified by an economical and practical three-step procedure monitored by AFB₁ conversion. And ESI-MS/MS analysis was done for identification of AFO. The following database searching (Protein Blast on NCBI) results did not show any homologous oxidase protein, which implied that AFO was mostly a new oxidase differing from other reported aflatoxin-converting enzymes such as fungal laccase and horse radish peroxidase. HPTLC analysis of the purified AFO activity suggested that the enzyme reacted at the bisfuran ring of AFB₁ which was the key toxic structure. Therefore, all these investigations implied a new choice for biodegradation of aflatoxin in foods and feeds with the practical application of AFO.     

Intralimb compensation strategy depends on the nature of joint perturbation in human hopping

Due to the well-described spring-mass dynamics of bouncing gaits, human hopping is a tractable model for elucidating basic neuromuscular compensation principles. We tested whether subjects would employ a multi-joint or single-joint response to stabilize leg stiffness while wearing a spring-loaded ankle-foot orthosis (AFO) that applied localized resistive and assistive torques to the ankle. We analyzed kinematics and kinetics data from nine subjects hopping in place on one leg, at three frequencies (2.2, 2.4, and 2.8Hz) and three orthosis conditions (freely articulating AFO, AFO with plantarflexion resistance, and AFO with plantarflexion assistance). Leg stiffness was invariant across AFO conditions, however, compensation strategy depended upon the nature of the applied load. Biological ankle stiffness increased in response to a resistive load at twice the rate that it decreased with an assitive load. Ankle adjustments alone fully compensated for an assistive load with no net change in combined (biological plus applied) total ankle stiffness (p > or =0.133). In contrast, a resistive load resulted in a 7.4-9.0% increase in total ankle stiffness across frequencies and a concomitant 10-15% increase in knee joint stiffness at each frequency (p< or =0.037). The increased knee joint stiffness in response to resistive ankle load allowed subjects to maintain a more flexed knee at mid-stance, which attenuated the effect of the increased total ankle joint stiffness to preserve leg stiffness and whole limb biomechanical performance. Our findings suggest humans maintain invariant leg stiffness in bouncing gaits through different intralimb compensation strategies that are specific to the nature of the joint loading.     

The effects of alignment of an articulated ankle-foot orthosis on lower limb joint kinematics and kinetics during gait in individuals post-stroke

Mechanical tuning of an ankle-foot orthosis (AFO) is important in improving gait in individuals post-stroke. Alignment and resistance are two factors that are tunable in articulated AFOs. The aim of this study was to investigate the effects of changing AFO ankle alignment on lower limb joint kinematics and kinetics with constant dorsiflexion and plantarflexion resistance in individuals post-stroke. Gait analysis was performed on 10 individuals post-stroke under four distinct alignment conditions using an articulated AFO with an ankle joint whose alignment is adjustable in the sagittal plane. Kinematic and kinetic data of lower limb joints were recorded using a Vicon 3-dimensional motion capture system and Bertec split-belt instrumented treadmill. The incremental changes in the alignment of the articulated AFO toward dorsiflexion angles significantly affected ankle and knee joint angles and knee joint moments while walking in individuals post-stroke. No significant differences were found in the hip joint parameters. The alignment of the articulated AFO was suggested to play an important role in improving knee joint kinematics and kinetics in stance through improvement of ankle joint kinematics while walking in individuals post-stroke. Future studies should investigate long-term effects of AFO alignment on gait in the community in individuals post-stroke.     

Effect of plantarflexion resistance of an ankle-foot orthosis on ankle and knee joint power during gait in individuals post-stroke

Plantarflexion resistance of an ankle-foot orthosis (AFO) plays an important role to prevent foot-drop, but its impact on push-off has not been well investigated in individuals post-stroke. The aim of this study was to investigate the effect of plantarflexion resistance of an articulated AFO on ankle and knee joint power of the limb wearing the AFO in individuals post-stroke. Gait analysis was performed on 10 individuals with chronic stroke using a Vicon 3-dimensional motion capture system and a Bertec split-belt instrumented treadmill. They walked on the treadmill under 4 plantarflexion resistance levels (S1 < S2<S3 < S4) set on the AFO with resistance adjustable ankle joints. The ankle and knee joint power calculations were performed using Visual3D, and mean values were plotted across a gait cycle. Statistical analyses revealed significant differences in the peak ankle joint power generation according to the plantarflexion resistance of the AFO (P = 0.008). No significant differences were found in the knee joint power. Peak ankle joint power generation [Median (IQR: Interquartile range)] were S1: 0.0517 (0.0238–0.1071) W/kg, S2: 0.0342 (0.0132–0.0862) W/kg, S3: 0.0353 (0.0127–0.0821) W/kg, and S4: 0.0234 (0.0087–0.06764) W/kg. Reduction of the peak ankle joint power generation appeared to be related to reduction in the peak plantarflexion angular velocity at late stance due to increases in the plantarflexion resistance of the AFO. This study showed that peak ankle joint power generation was significantly, and somewhat systematically, affected by plantarflexion resistance of the AFO in individuals post-stroke.     

Theoretical prediction of the gel electrophoretic retardation changes due to point mutations in a tract of SV40 DNA.

The changes of gel electrophoretic retardation due to single base substitutions in a 173 bp fragment of Sv40 DNA were predicted by using a theoretical model based on conformational energy calculations. As described in previous papers, this model allows successful prediction of the gel electrophoretic retardation of synthetic as well as natural DNAs reported in literature. The experimental retardations related to 195 point-mutated DNAs were reproduced with a standard deviation of 0.05 comparable with the experimental one of 0.04. This result, which represents a very critical test for the proposed model, indicates that DNA superstructures can be satisfactorily predicted on the simple physical basis of the integration of the nearest-neighbour perturbations in the dinucleotide steps. Thus, cooperative effects appear, in the majority of cases investigated, to play a second order role.     

A simple model of a steady state differentiating cell system

A simple theoretical model is hypothesized to describe the steady state behavior of a differentiating cell system as exemplified by blood cells. The cell system consists of several morphologically distinguishable cell classes which develop sequentially. Each cell class except the last one is mitotically capable. Mitosis is assumed to be either heteromorphogenic, homomorphogenic, or asymmetric. Some algebraic equations are derived which are conservation equations describing the flux of cells from one class to another. The theoretical considerations have been applied to some experimental observations in humans concerning neutrophil production, particularly in reference to relative cell numbers and mitotic fractions of the myeloblast, promyelocyte, and myelocyte cell classes. These observations are utilized to help determine the values of the parameters which characterize the model. Among these parameters are the generation times of the various cell classes, and the predicted values of the generation times are found to be in excellent agreement with observed grain-count halving times. However, the predicted mitotic times are in disagreement with their observed values.     

Engineering a thermostable protein via optimization of charge-charge interactions on the protein surface

A simple theoretical model for increasing the protein stability by adequately redesigning the distribution of charged residues on the surface of the native protein was tested experimentally. Using the molecule of ubiquitin as a model system, we predicted possible amino acid substitutions on the surface of this protein which would lead to an increase in its stability. Experimental validation for this prediction was achieved by measuring the stabilities of single-site-substituted ubiquitin variants using urea-induced unfolding monitored by far-UV CD spectroscopy. We show that the generated variants of ubiquitin are indeed more stable than the wild-type protein, in qualitative agreement with the theoretical prediction. As a positive control, theoretical predictions for destabilizing amino acid substitutions on the surface of the ubiquitin molecule were considered as well. These predictions were also tested experimentally using correspondingly designed variants of ubiquitin. We found that these variants are less stable than the wild-type protein, again in agreement with the theoretical prediction. These observations provide guidelines for rational design of more stable proteins and suggest a possible mechanism of structural stability of proteins from thermophilic organisms.     

A novel device to evaluate the stiffness of ankle-foot orthosis devices

The evaluation of mechanical behavior of plastic Ankle-Foot Orthosis (AFO) is important since AFO can provide an efficient support to patients with disabilities in locomotion. This paper reports on a novel testing apparatus that allows: (a) the evaluation of AFO stiffness in sagittal and frontal planes; (b) the conduction of semi-automatic trials; and, finally, (c) a global accuracy associated to the AFO stiffness values always less than 4%. The stiffness values are determined by the measurements of the imposed relative displacements between the foot and the shank of the orthosis and the induced reaction forces. The data collected together in an exact 2-D approach, together with those provided by gait analysis systems, allows to better understand gait alteration induced by ankle orthosis, and to improve clinical management of patients.     

Computer Simulation of After-Inhibition in Crayfish Slowly Adapting Stretch Receptor Neuron

A theoretical model is described which is able to mimic the responses of slowly adapting stretch receptor neurons of crayfish to applied currents. Its principal feature is postspike inhibition, in which each nerve impulses produces a small inhibitory current that decays with a simple exponential time-course that is long compared with normal interspike intervals. The model was simulated with both an analogue and a digital computer. Parameters for particular model neurons were determined both by an analysis of experimental data obtained from adaptation to constant injected currents, and by matching computer output to the data. Parameter values estimated by the two techniques agreed within ±10%. Model parameters determined from adaptation data successfully predicted the magnitude and time-course of posttetanic hyperpolarization (PTH) in the stretch receptor neuron. In addition, model neurons were able to reproduce posttetanic depression (PTD) as seen in stretch receptor cells.     

Impact of ankle foot orthosis stiffness on Achilles tendon and gastrocnemius function during unimpaired gait

Ankle foot orthoses (AFOs) are designed to improve gait for individuals with neuromuscular conditions and have also been used to reduce energy costs of walking for unimpaired individuals. AFOs influence joint motion and metabolic cost, but how they impact muscle function remains unclear. This study investigated the impact of different stiffness AFOs on medial gastrocnemius muscle (MG) and Achilles tendon (AT) function during two walking speeds. We performed gait analyses for eight unimpaired individuals. Each individual walked at slow and very slow speeds with a 3D printed AFO with no resistance (free hinge condition) and four levels of ankle dorsiflexion stiffness: 0.25 Nm/°, 1 Nm/°, 2 Nm/°, and 3.7 Nm/°. Motion capture, ultrasound, and musculoskeletal modeling were used to quantify MG and AT lengths with each AFO condition. Increasing AFO stiffness increased peak AFO dorsiflexion moment with decreased peak knee extension and peak ankle dorsiflexion angles. Overall musculotendon length and peak AT length decreased, while peak MG length increased with increasing AFO stiffness. Peak MG activity, length, and velocity significantly decreased with slower walking speed. This study provides experimental evidence of the impact of AFO stiffness and walking speed on joint kinematics and musculotendon function. These methods can provide insight to improve AFO designs and optimize musculotendon function for rehabilitation, performance, or other goals.     

Oxidase or peptidase? A computational insight into a putative aflatoxin oxidase from Armillariella tabescens

Aflatoxin oxidase (AFO), an enzyme isolated from Armillariella tabescens, has been reported to degrade aflatoxin B1 (AFB1). However, recent studies reported sequence and structure similarities with the dipeptidyl peptidase III (DPP III) family of enzymes and confirmed peptidase activity toward DPP III substrates. In light of these investigations, an extensive computational study was performed in order to improve understanding of the AFO functions. Steered MD simulations revealed long-range domain motions described as protein opening, characteristic for DPPs III and necessary for substrate binding. Newly identified open and partially open forms of the enzyme closely resemble those of the human DPP III orthologue. Docking of a synthetic DPP III substrate Arg₂-2-naphthylamide revealed a binding mode similar to the one found in crystal structures of human DPP III complexes with peptides with the S1 and S2 subsites’ amino acid residues conserved. On the other hand, no energetically favorable AFB1 binding mode was detected, suggesting that aflatoxins are not good substrates of AFO. High plasticity of the zinc ion coordination sphere within the active site, consistent with that of up to date studied DPPs III, was observed as well. A detailed electrostatic analysis of the active site revealed a predominance of negatively charged regions, unsuitable for the binding of the neutral AFB1. The present study is in line with the most recent experimental study on this enzyme, both suggesting that AFO is a typical member of the DPP III family.     

Simulating the Entropic Collapse of Coarse-Grained Chromosomes

Depletion forces play a role in the compaction and decompaction of chromosomal material in simple cells, but it has remained debatable whether they are sufficient to account for chromosomal collapse. We present coarse-grained molecular dynamics simulations, which reveal that depletion-induced attraction is sufficient to cause the collapse of a flexible chain of large structural monomers immersed in a bath of smaller depletants. These simulations use an explicit coarse-grained computational model that treats both the supercoiled DNA structural monomers and the smaller protein crowding agents as combinatorial, truncated Lennard-Jones spheres. By presenting a simple theoretical model, we quantitatively cast the action of depletants on supercoiled bacterial DNA as an effective solvent quality. The rapid collapse of the simulated flexible chromosome at the predicted volume fraction of depletants is a continuous phase transition. Additional physical effects to such simple chromosome models, such as enthalpic interactions between structural monomers or chain rigidity, are required if the collapse is to be a first-order phase transition.     

Reconciling the optimal and empirical approaches to modelling stomatal conductance

Models of vegetation function are widely used to predict the effects of climate change on carbon, water and nutrient cycles of terrestrial ecosystems, and their feedbacks to climate. Stomatal conductance, the process that governs plant water use and carbon uptake, is fundamental to such models. In this paper, we reconcile two long‐standing theories of stomatal conductance. The empirical approach, which is most commonly used in vegetation models, is phenomenological, based on experimental observations of stomatal behaviour in response to environmental conditions. The optimal approach is based on the theoretical argument that stomata should act to minimize the amount of water used per unit carbon gained. We reconcile these two approaches by showing that the theory of optimal stomatal conductance can be used to derive a model of stomatal conductance that is closely analogous to the empirical models. Consequently, we obtain a unified stomatal model which has a similar form to existing empirical models, but which now provides a theoretical interpretation for model parameter values. The key model parameter, g₁, is predicted to increase with growth temperature and with the marginal water cost of carbon gain. The new model is fitted to a range of datasets ranging from tropical to boreal trees. The parameter g₁ is shown to vary with growth temperature, as predicted, and also with plant functional type. The model is shown to correctly capture responses of stomatal conductance to changing atmospheric CO₂, and thus can be used to test for stomatal acclimation to elevated CO₂. The reconciliation of the optimal and empirical approaches to modelling stomatal conductance is important for global change biology because it provides a simple theoretical framework for analyzing, and simulating, the coupling between carbon and water cycles under environmental change.     

Calorimetric investigation of aerobic fermentations

A modified bench scale calorimeter has been employed to determine the heat generated by various microbial strains growing on a range of different substrates, covering degrees of reduction from 3 to 6.13. The results are analyzed, and interpreted in the light of coupled enthalpy and elemental balances. The heat released by the microbial cultures has been found to correlate linearly with other process variables, such as biomass generation and oxygen uptake. The ratio between the heat generated and the biomass formed, the so-called "heat yield" (Y(Q/x)), has been shown both on theoretical and experimental grounds to increase with increasing degree of reduction of the substrate and to decrease with increasing biomass yield. The two effects could be combined into a simple model which permits the amount of heat released per unit of biomass formed to be predicted from the degree of reduction of the substrate as the only independent variable. The ratio between the heat generated and the oxygen taken up was constant at 440 kJ (mol O(2))(-1) throughout all experiments as expected from theoretical considerations for strongly aerobic processes.     

Topological analysis of the Rhodobacter capsulatus PucC protein and effects of C-terminal deletions on light-harvesting complex II.

A theoretical model for the cytoplasmic membrane topology of the Rhodobacter capsulatus PucC protein was derived and tested experimentally with pucC'::pho'A gene fusions. The alkaline phosphatase (AP) activities of selected fusions were assayed, and the resultant pattern of high and low activity was compared with that of the theoretical model. High AP activity correlated well with fusion joints located in regions predicted to be periplasmic, and most fusions in predicted cytoplasmic loops yield approximately 1/20th as much activity. Replacement of pho'A with lac'Z in nine of the fusions confirmed the topology, as beta-galactosidase activities were generally reciprocal to the corresponding AP activity. On the basis of the theoretical analysis and the information provided by the activities of fusions, a model for PucC topology in which there are 12 membrane-spanning segments and both the N and C termini are located in the cytoplasm is proposed. Translationally out-of-frame pucC::phoA fusions were expressed in an R. capsulatus delta pucC strain. None of the fusions missing only one or two of the proposed C-terminal transmembrane segments restored the wild-type phenotype, suggesting that the C terminus of PucC is important for function.     

Pseudophosphorylation of the smooth muscle 20 000 dalton myosin light chain. An artifact due to protein modification

The use of isoelectric focusing as a technique for quantifying the stoichiometry of phosphorylation of the 20 kDa smooth muscle myosin light chain (LC20) was found to overestimate true levels of phosphorylation under certain conditions due to the occurrence of LC20 charge modification. Modification of unphosphorylated LC20 produced a band of 'pseudophosphorylated' LC20 which co-focused with phosphorylated LC20. LC20 modification was found to occur when samples were subjected to electrophoresis under nonreducing conditions in the presence of ammonium persulfate. The overestimation of LC20 phosphorylation due to pseudophosphorylation was examined for both purified myosin and extracts from contracting smooth muscle and found to be greatest at low levels of LC20 phosphorylation. A simple theoretical model was developed which accurately predicted the effects of charge modification on the measured level of phosphorylation. LC20 modification was shown to be completely eliminated by the inclusion of dithiothreitol in extraction buffers and the pre-electrophoresis of sodium thioglycolate into gels.     

牙鲆钙调素基因在原核和真核细胞中的表达

设计特异引物,以SMART cDNA为模板,应用PCR方法扩增牙鲆钙调素基因（Paralichthys olivocew calmodulin,PoCaM）。计算机辅助分析表明,PoCaM基因编码149个氨基酸的推定蛋白,其分子量为17kD,等电点为3．93,含有4个螺旋-环-螺旋样结构,与其它鱼类CaM氨基酸一致性为97．3％-100％。构建原核表达重组质粒pET32a／PoCaM,转化大肠杆菌B121（DE3）,用IPTG进行诱导表达,经SDS—PAGE蛋白电泳,结果显示PoCaM在大肠杆菌中进行了特异性融合表达,融合蛋白分子量约为34kD,与预期分子量大小一致。同时,以绿色荧光蛋白（GFP）为表达标签,构建真核表达重组质粒pEGFP—N3／PoCaM,经Lipofectamine 2000介导转染鲤鱼上皮瘤细胞（ Epithelioma papulosum cyprinid, EPC）,荧光显微镜观察显示,PoCaM在EPC细胞中进行瞬时表达,主要分布于细胞核及胞浆中[动物学报54（6）：1061—1067,2008]。     

Sub-exponential growth and coexistence of non-enzymatically replicating templates

The experimentally reported kinetic behaviour (sub-exponential but supra-linear growth) of non-enzymatic template replication is incorporated into a simple model of template competition. Sub-exponential growth is shown to lead to coexistence invariably. Thus coexistence of different non-enzymatically replicating sequences is predicted. This type of coexistence could have been important in maintaining a sufficient diversity of RNA modules used later to build functional molecules such as ribozymes. Experimental tests of this theoretical prediction are possible.