不同上样方式对蛋白质组双向电泳图谱质量的影响比较

蛋白质组技术难点之一是如何获取尽可能多的细胞或组织的蛋白信息.双向电泳蛋白斑点数目直接反映了实验蛋白质组信息的完整性.除样品制备外,蛋白上样方式对双向电泳图谱的质量和完整性有直接的影响.实验论文从以下3个方面考察不同的蛋白上样方式对双向电泳图谱的影响;即：水化上样与杯上样;一次上样与重复上样;以及酸性端加样与碱性端上样.实验结果发现;蛋白上样量较大时,杯上样方式的图谱斑点数目较水化上样方式明显增多;样品蛋白浓度较高时,稀释多次上样明显优于一次性浓缩蛋白上样;蛋白裂解液(MCF7乳腺癌细胞)在酸性端加样对偏碱性蛋白的分离未发现明显优势.相反,在等电聚焦伏小时(Vh) 足够的前提下,碱性端加样对偏碱性端蛋白反而有利,表现为斑点数目较多,而且等电点方向拖尾减轻.实验结果对提高双向电泳的质量以及相关蛋白质组信息的完整性提供了有益的技术参考.     

Deprivation and Enrichment in Laboratory Animal Environments

Resistance in the horse to trailer loading is a common source of stress and injury to horses and their handlers. The objective of this study was to determine whether nonaversive training based on the Tellington-Touch Equine Awareness Method (TTEAM; Tellington-Jones &; Bruns, 1988) would decrease loading time and reduce stress during loading for horses with a history of reluctance to load. Ten horses described by their owners as "problem loaders" were subjected to pretraining and posttraining assessments of loading. Each assessment involved two 7-min loading attempts during which heart rate and saliva cortisol were measured. The training consisted of six 30-min sessions over a 2-week period during which the horse and owner participated in basic leading exercises with obstacles simulating aspects of trailering. Assessment showed heart rate and saliva cortisol increased significantly during loading as compared to baseline (p < .001 and p < .05, respectively). Reassessment after training showed a decrease in loading time (p < .02), reduced heart rate during loading (p < .002), and reduced saliva cortisol as compared to pretraining assessments. Seven "good loaders" also were subject to loading assessment for physiological comparison. Increases in heart rate during loading were significantly higher in the good loaders (p < .001). Nonaversive training simulating aspects of loading may effectively reduce loading time and stress during loading for horses with a history of resistance to trailer loading.     

Trailer loading stress in horses: behavioral and physiological effects of nonaversive training (TTEAM)

Resistance in the horse to trailer loading is a common source of stress and injury to horses and their handlers. The objective of this study was to determine whether nonaversive training based on the Tellington-Touch Equine Awareness Method (TTEAM; Tellington-Jones &Bruns, 1988) would decrease loading time and reduce stress during loading for horses with a history of reluctance to load. Ten horses described by their owners as "problem loaders" were subjected to pretraining and posttraining assessments of loading. Each assessment involved two 7-min loading attempts during which heart rate and saliva cortisol were measured. The training consisted of six 30-min sessions over a 2-week period during which the horse and owner participated in basic leading exercises with obstacles simulating aspects of trailering. Assessment showed heart rate and saliva cortisol increased significantly during loading as compared to baseline (p <.001 and p <.05, respectively). Reassessment after training showed a decrease in loading time (p <.02), reduced heart rate during loading (p <.002), and reduced saliva cortisol as compared to pretraining assessments. Seven "good loaders" also were subject to loading assessment for physiological comparison. Increases in heart rate during loading were significantly higher in the good loaders (p <.001). Nonaversive training simulating aspects of loading may effectively reduce loading time and stress during loading for horses with a history of resistance to trailer loading.     

The effect of altered loading following rotator cuff tears in a rat model on the regional mechanical properties of the long head of the biceps tendon

Biceps tendon pathology is a common clinical problem often seen in conjunction with rotator cuff tears. A previous study found detrimental changes to biceps tendons in the presence of rotator cuff tears in a rat model. Therefore, the objective of this study was to utilize this model along with models of altered loading to investigate the effect of altered loading on the initiation of these detrimental changes. We created supraspinatus and infraspinatus rotator cuff tears in the rat and followed these tears with either increased or decreased loading. Mechanical properties were determined along the length of the biceps tendon 4 and 8 weeks following injury. At the insertion site, stiffness increased with decreased loading, while detrimental changes were seen with increased loading 4 weeks following detachments. Increased loading resulted in decreased mechanical properties along the entire tendon length at both time points. Decreased loading resulted in both increased and decreased tendon properties at different regions of the tendon at 4 weeks, but by 8 weeks, there were no differences between decreased loading and detachment alone. We could not conclude where changes begin in the tendon with altered loading, but did demonstrate that regional differences exist. These results support that there is an effect of altered loading, as decreased loading resulted in variable changes at 4 weeks that were no different from detachment alone by 8 weeks, and increased loading resulted in detrimental properties along the entire length at both 4 and 8 weeks.     

Mechanical loading of orthodontic miniscrews - significance and problems: an experimental study

Orthodontic miniscrews are exposed to three mechanical loading phases during clinical use: torsional loading upon insertion, flexural loading during anchorage function, and torsional loading upon removal. The aim of this study was to simulate clinical loading conditions for different types of orthodontic miniscrews in vitro to quantify the effects of combined torsional and bending stress. Various orthodontic miniscrew systems (Lomas, Dual-top, Aarhus anchorage, Tomas-pin and T.I.T.A.N.-pin) comprising 10 samples each were subjected to the following loading sequences in vitro: a torsional load corresponding to manual insertion with limited torque; and flexural loading at two different insertion depths. For all screw systems with torsional pre-loading (simulating insertion), subsequent flexural loading (simulating anchorage) yielded permanent deformations of approximately 0.15-0.25 mm, depending on the insertion depth. Since EDX analysis revealed comparable elemental compositions for the different screw systems, the differences in mechanical properties are attributed to screw design. Torsional loading during screw insertion may cause premature mechanical weakening and needs to be minimized. Unless fully inserted, screws show pronounced plastic deformation and hence fracture risk under subsequent flexural loading.     

Femoral stiffness and strength critically depend on loading angle: a parametric study in a mouse-inbred strain

Biomechanical tests of human femora have shown that small variations of the loading direction result in significant changes in measured bone mechanical properties. However, the heterogeneity in geometrical and bone tissue properties does not make human bones well suited to reproducibly assess the effects of loading direction on stiffness and strength. To precisely quantify the influence of loading direction on stiffness and strength of femora loaded at the femoral head, we tested femora from C57BL/6 inbred mice. We developed an image-based alignment protocol and investigated the loading direction influence on proximal femur stiffness and strength. An aluminum femoral phantom and C57BL/6 femora were tested under compression with different loading directions. Both tests, with the aluminum phantom and the murine bones, showed and quantified the linear dependence of stiffness on loading direction: a 5 degrees change in loading direction resulted in almost 30% change in stiffness. Murine bone testing also revealed and quantified the variation in strength due to loading direction: 5 degrees change in loading direction resulted in 8.5% change in strength. In conclusion, this study quantified, for the first time, the influence of misalignment on bone stiffness and strength for femoral head loading. We showed the extreme sensitivity of this site regarding loading direction.     

Finite element analysis of the effect of loading curve shape on brain injury predictors

Prediction of traumatic and mild traumatic brain injury is an important factor in managing their prevention. Currently, the prediction of these injuries is limited to peak linear and angular acceleration loading curves derived from laboratory reconstructions. However it remains unclear as to what aspect of these loading curves contributes to brain tissue damage. This research uses the University College Dublin Brain Trauma Model (UCDBTM) to analyse three distinct loading curve shapes meant to represent different helmet loading scenarios. The loading curves were applied independently in each axis of linear and angular acceleration and their effect on currently used predictors of TBI and mTBI was examined. Loading curve shape A had a late time to peak, B an early time to peak and C had a consistent plateau. The areas under the curve for all three loading curve shapes were identical. The results indicate that loading curve A produced consistently higher maximum principal strains and Von Mises stress than the other two loading curve types. Loading curve C consistently produced the lowest values of maximum principal strain and Von Mises stress, with loading curve B being lowest in only 2 cases. The areas of peak Von Mises stress and Principal strain also varied depending on loading curve shape and acceleration input.     

Bone morphology allows estimation of loading history in a murine model of bone adaptation

Bone adapts its morphology (density/micro- architecture) in response to the local loading conditions in such a way that a uniform tissue loading is achieved (‘Wolff’s law’). This paradigm has been used as a basis for bone remodeling simulations to predict the formation and adaptation of trabecular bone. However, in order to predict bone architectural changes in patients, the physiological external loading conditions, to which the bone was adapted, need to be determined. In the present study, we developed a novel bone loading estimation method to predict such external loading conditions by calculating the loading history that produces the most uniform bone tissue loading. We applied this method to murine caudal vertebrae of two groups that were in vivo loaded by either 0 or 8 N, respectively. Plausible load cases were sequentially applied to micro-finite element models of the mice vertebrae, and scaling factors were calculated for each load case to derive the most uniform tissue strain-energy density when all scaled load cases are applied simultaneously. The bone loading estimation method was able to predict the difference in loading history of the two groups and the correct load magnitude for the loaded group. This result suggests that the bone loading history can be estimated from its morphology and that such a method could be useful for predicting the loading history for bone remodeling studies or at sites where measurements are difficult, as in bone in vivo or fossil bones.     

Chondrocyte intracellular calcium, cytoskeletal organization, and gene expression responses to dynamic osmotic loading

While chondrocytes in articular cartilage experience dynamic stimuli from joint loading activities, few studies have examined the effects of dynamic osmotic loading on their signaling and biosynthetic activities. We hypothesize that dynamic osmotic loading modulates chondrocyte signaling and gene expression differently than static osmotic loading. With the use of a novel microfluidic device developed in our laboratory, dynamic hypotonic loading (–200 mosM) was applied up to 0.1 Hz and chondrocyte calcium signaling, cytoskeleton organization, and gene expression responses were examined. Chondrocytes exhibited decreasing volume and calcium responses with increasing loading frequency. Phalloidin staining showed osmotic loading-induced changes to the actin cytoskeleton in chondrocytes. Real-time PCR analysis revealed a stimulatory effect of dynamic osmotic loading compared with static osmotic loading. These studies illustrate the utility of the microfluidic device in cell signaling investigations, and their potential role in helping to elucidate mechanisms that mediate chondrocyte mechanotransduction to dynamic stimuli. cartilage; calcium signaling; actin cytoskeleton; aggrecan     

Effects of mechanical loading and hypercapnia on inspiratory muscle EMG.

The electromyograms of the diaphragm and an external intercostal muscle were analyzed to see if the effects of hypercapnia on inspiratory muscle electrical activity could be distinguished from those of mechanical loading and to determine whether changes in inspiratory muscle electrical activity were a sueful measure of CO2 response during mechanical loading. Anesthetized dogs were studied: 1) during progressive hypercapnia without mechanical loading, 2) during flow-resistive and elastic loading at constant PCO2, and 3) during progressive hypercapnia and mechanical loading. Both mechanical loading and hypercapnia increased total inspiratory diaphragmatic and intercostal muscle electrical activity. However, inspiratory duration was increased by mechanical loads but reduced by hypercapnia. Because of these changes in inspiratory duration, the average rate of diaphragmatic electrical activity remained unaffected by mechanical loading before and after vagotomy but was increased by hypercapnia. In contrast, both hypercapnia and mechanical loading increased the average rate of intercostal muscle electrical activity. There was a greater increase in both total and average rate of intercostal muscle electrical activity during hypercapnia in the presence of mechanical loading than during unloaded breathing. However, the change in total and average rate of diaphragmatic electrical activity with PCO2 was unaffected by added mechanical loads. These results suggest that diaphragmatic but not intercostal muscle electrical activity can be used as an index of CO2 response even during mechanical loading.     

Electroporation of human embryonic stem cells: Small and macromolecule loading and DNA transfection

Cryopreservation, directed differentiation, and genetic manipulation of human embryonic stem cells (hESCs) all require the transport of exogenous small molecules, proteins, or DNA into the cell. The absence of standard small and macromolecule loading techniques in hESCs as well as the inadequacies of current DNA transfection techniques have led us to develop electroporation as an efficient loading and transfection methodology. The electroporation parameters of pulse voltage, duration, and number have been explored and evaluated in terms of cell viability, molecular loading, and transfection efficiency on a per cell basis. Small molecule loading was assessed using propidium iodide (PI) and the disaccharide trehalose. Additionally, protein loading was investigated using a glutathione-S-transferase green fluorescent protein (GST-GFP) conjugate, and DNA transfection optimization was performed by constitutive expression of GFP from a plasmid. The optimum pulse voltage must balance cell viability, which decreases as voltage increases, and loading efficiency, which increases at higher voltages. Short pulse times of 0.05 ms facilitated PI and trehalose loading, whereas 0.5 ms or more was required for GST-GFP loading and DNA transfection. Multiple pulses increased per cell loading of all molecules, though there was a dramatic loss of viability with GST-GFP loading and DNA transfection, likely resulting from the longer pulse duration required to load these molecules.     

Effect of dynamic loading on the frictional response of bovine articular cartilage

The objective of this study was to test the hypotheses that (1) the steady-state friction coefficient of articular cartilage is significantly smaller under cyclical compressive loading than the equilibrium friction coefficient under static loading, and decreases as a function of loading frequency; (2) the steady-state cartilage interstitial fluid load support remains significantly greater than zero under cyclical compressive loading and increases as a function of loading frequency. Unconfined compression tests with sliding of bovine shoulder cartilage against glass in saline were carried out on fresh cylindrical plugs (n=12), under three sinusoidal loading frequencies (0.05, 0.5 and 1 Hz) and under static loading; the time-dependent friction coefficient mu(eff) was measured. The interstitial fluid load support was also predicted theoretically. Under static loading mu(eff) increased from a minimum value (mu(min)=0.005+/-0.003) to an equilibrium value (mu(eq)=0.153+/-0.032). In cyclical compressive loading tests mu(eff) similarly rose from a minimum value (mu(min)=0.004+/-0.002, 0.003+/-0.001 and 0.003+/-0.001 at 0.05, 0.5 and 1 Hz) and reached a steady-state response oscillating between a lower-bound (mu(lb)=0.092+/-0.016, 0.083+/-0.019 and 0.084+/-0.020) and upper bound (mu(ub)=0.382+/-0.057, 0.358+/-0.059, and 0.298+/-0.061). For all frequencies it was found that mu(ub)>mu(eq) and mu(lb)相似文献   

Computer simulation of the effects of shoe cushioning on internal and external loading during running impacts

Biomechanical aspects of running injuries are often inferred from external loading measurements. However, previous research has suggested that relationships between external loading and potential injury-inducing internal loads can be complex and nonintuitive. Further, the loading response to training interventions can vary widely between subjects. In this study, we use a subject-specific computer simulation approach to estimate internal and external loading of the distal tibia during the impact phase for two runners when running in shoes with different midsole cushioning parameters. The results suggest that: (1) changes in tibial loading induced by footwear are not reflected by changes in ground reaction force (GRF) magnitudes; (2) the GRF loading rate is a better surrogate measure of tibial loading and stress fracture risk than the GRF magnitude; and (3) averaging results across groups may potentially mask differential responses to training interventions between individuals.     

On the conditional equivalence of chemical loading and mechanical loading on articular cartilage

Osmotic pressure loading of articular cartilage has been customarily invoked to be equivalent to mechanical loading. In the literature, this equivalence is defined by the amount of water squeezed from the tissue, i.e. if the amount of water content lost by these two modes of loading are the same, it has been generally regarded that the two loadings are equivalent. This assumption has never been proven. Using the water content lost concept, in this paper, we derived the exact conditions under which an osmotic pressure loading of cartilage can be considered to be equivalent to a mechanical loading. However, the mechanical loading condition satisfying this equivalancy criterion, i.e. an isotropic loading delivered via a porous–permeable rigid platen uniformily applied all around the specimen, is not practically achievable. Moreover, even if this were achieved experimentally, the interstitial fluid pressure caused by the two loading conditions are not the same. This result has important ramifications for interpretation of experimental data from mechanical stimulations of cartilage explant studies.     

An intra-oral hydraulic system for controlled loading of dental implants

This study reports a method for controlling loads on an in vivo dental implant and its application for the investigation of early loading versus delayed loading of dental implants. The method was developed for the purpose of studying an ongoing hypothesis that amounts to bone loss around dental implants are related to mechanical-mediated adaptation of the alveolar bone. Using a customized intra-oral hydraulic system, the daily loading over a dental implant has been completed and recorded for six Sinclair swine. Each pig had a 5-month duration implant loading. During the experiments (loading), no analgesic treatment was supplied. The mean of the in vivo daily loadings was confirmed through an in vitro bench test after each animal was euthanized. Variations of the averaged loading input among the six animals were smaller than 10%. Preliminary data produced by the model suggests that cervical bone loss is less for early loading than for delayed loading. The current system is expected to provide a useful load control model for the study of alveolar bone adaptation around dental implants in relation to various loadings.     

Bone formation induced by a novel form of mechanical loading on joint tissue.

Because of insufficient mechanical loading, exposure to weightlessness in space flight reduces bone mass. In order to maintain bone mass in a weightless condition, we investigated a novel form of mechanical loading--joint loading. Since some part of gravity-induced loading to our skeletal system is absorbed by viscoelastic deformation of joint tissues, we hypothesized that deformation of joint tissues would generate fluid flow in bone and stimulate bone formation in diaphyseal cortical bone. In order to test the hypothesis, we applied directly oscillatory loading to an elbow joint of mice and conducted bone histomorphometry on the diaphysis of ulnae. Using murine femurs ex vivo, streaming potentials were measured to evaluate a fluid flow induced by joint loading. Bone histomorphometry revealed that compared to no loading control, elbow loading increased mineralizing surface, mineral apposition rate, and bone formation rate 3.2-fold, 3.0-fold, and 7.9-fold, respectively. We demonstrated that joint loading generated a streaming potential in a medullar cavity of femurs. The results support a novel mechanism, in which joint loading stimulates effectively bone formation possibly by generating fluid flow, and suggest that a supportive attachment to joints, driven passively or actively, would be useful to maintain bone mass of astronauts during an exposure to weightlessness.     

Intrapulmonary Co2 receptors and ventilatory response to lung Co2 loading

The possible role of intrapulmonary CO2 receptors (IPC) in arterial CO2 partial pressure (PaCO2) homeostasis was investigated by comparing the arterial blood gas and ventilatory responses to CO2 loading via the inspired gas and via the venous blood. Adult male Pekin ducks were decerebrated 1 wk prior to an experiment. Venous CO2 loading was accomplished with a venovenous extracorporeal blood circuit that included a silicone-membrane blood oxygenator. The protocol randomized four states: control (no loading), venous CO2 loading, inspired CO2 loading, and venous CO2 unloading. Intravenous and inspired loading both resulted in hypercapnic hyperpnea. Comparison of the ventilatory sensitivity (delta VE/delta PaCO2) showed no significant difference between the two loading regimes. Likewise, venous CO2 unloading led to a significant hypocapnic hypopnea. Sensitivity to changes in PaCO2 could explain the response of ventilation under these conditions. The ventilatory pattern, however, was differentially sensitive to the route of CO2 loading; inspired CO2 resulted in slower deeper breathing than venous loading. It is concluded that IPC play a minor role in adjusting ventilation to match changes in pulmonary CO2 flux but rather are involved in pattern determination.     

Ecophysiology of phloem loading in source leaves

The nature of phloem loading of photosynthesis products – either symplastic or apoplastic – has been a matter of debate over the last two decades. This controversy was reconciled by proposing a multiprogrammed loading mechanism. Different modes of phloem loading were distinguished on the basis of the variety of plasmodesmatal connectivity between the minor vein elements. Physiological evidence for at least two phloem loading mechanisms as well as recent support for coincidence between plasmodesmatal connectivity and the loading mechanism is shortly reviewed. The present paper attempts to correlate the plasmodesmatal connectivity between sieve element/companion cell complex and the adjacent cells (the minor vein configuration) – and implicitly the associate phloem loading mechanisms – with different types of climate. The minor vein configuration is a family characteristic. This enables one to relate vein configuration with ecosystem using the family distribution over the globe. The uneven distribution of vein types between terrestrial ecosystems indicates that apoplastic phloem loading predominates in cold and dry climate zones. Projection of the minor vein configuration on the Takhtajan system of flowering plants suggests evolution from apoplastic to symplastic phloem loading. Accordingly, the distribution of minor vein configurations suggests that drought and temperature stress have led to the transformation of the ancient symplastic mode into the more advanced apoplastic mode of loading.     

Iron loading into ferritin can be stimulated or inhibited by the presence of cations and anions: a specific role for phosphate

Phosphate and other oxo-anions have been shown to stimulate the rate of iron loading into ferritin (J. Polanams, A.D. Ray, R.K. Watt, Inorg. Chem. 44 (2005) 3203-3209). This study was undertaken to determine if accelerated iron loading was a specific effect for phosphate and closely associated oxo-anions or if it was a general anion effect. Controls were also performed with mono-valent cations to determine the effect of these cations on iron loading into ferritin. Cations were shown to slow the rate of iron loading into ferritin. Fluoride and iodide were shown to slow the iron loading process of ferritin. Sulfate was also shown to slow iron loading into ferritin to a more significant extent than the cations or halides tested. The trigonal planar oxo-anions, carbonate and nitrate, did not inhibit or stimulate iron loading. We conclude that the increased rate of iron loading into ferritin is specific to phosphate and other closely associated tetrahedral oxo-anion analogs, that the effect is driven by the insolubility of the iron and anion complex, and that in general, cations and anions slow the rate of iron loading into ferritin.