Membrane electrical parameters in turtle bladder measured using impedance-analysis techniques

Summary Equivalent-circuit impedance analysis experiments were performed on the urinary bladders of freshwater turtles in order to quantify membrane ionic conductances and areas, and to investigate how changes in these parameters are associated with changes in the rate of proton secretion in this tissue. In all experiments, sodium reabsorption was inhibited thereby unmasking the electrogenic proton secretion process. We report the following: (1) transepithelial impedance is represented exceptionally well by a simple equivalent-circuit model, which results in estimates of the apical and basolateral membrane ionic conductances and capacitances; (2) when sodium transport is inhibited with mucosal amiloride and serosal ouabain, the apical and basolateral membrane conductances and capacitances exhibit a continual decline with time; (3) this decline in the membrane parameters is most likely caused by subtle time-dependent changes in cell volume, resulting in changes in the areas of the apical and basolateral membranes; (4) stable membrane parameters are obtained if the tissue is not treated with ouabain, and if the oncotic pressure of the serosal solution is increased by the addition of 2% albumin; (5) inhibition of proton secretion using acetazolamide in CO₂ and HCO ₃ ^– -free bathing solutions results in a decrease in the area of the apical membrane, with no significant change in its specific conductance; (6) stimulation of proton transport with CO₂ and HCO ₃ ^– -containing serosal solution results in an increase in the apical membrane area and specific conductance. These results show that our methods can be used to measure changes in the membrane electrophysiological parameters that are related to changes in the rate of proton transport. Notably, they can be used to quantify in the live tissue, changes in membrane area resulting from changes in the net rates of endocytosis and exocytosis which are postulated to be intimately involved in the regulation of proton transport.     

Histological and mechanical evaluation of antifreeze peptide (Afp1m) cryopreserved skin grafts post transplantation in a rat model

The objective of this study was to evaluate the use of Afp1m as a cryopreservative agent for skin by examining the transplanted skin histological architecture and mechanical properties following subzero cryopreservation. Thirty four (34) rats with an average weight of 208 ± 31 g (mean ± SD), were used. Twenty four (n = 24) rats were equally divided into four groups: (i) immediate non-cryopreserved skin autografts (onto same site), (ii) immediate non-cryopreserved skin autografts (onto different sites), (iii) skin autografts cryopreserved with glycerol for 72 h and (iv) skin autografts cryopreserved with Afp1m for 72 h at −4 °C. Rounded shaped full-thickness 1.5–2.5 cm in diameter skin was excised from backs of rats for the autograft transplantation. Non-cryopreserved or cryopreserved auto skin graft were positioned onto the wound defects and stitched. Non-transplanted cryopreserved and non-cryopreserved skin strips from other ten rats (n = 10) were allowed for comparative biomechanical test. All skin grafts were subjected to histological and mechanical examinations at the end of day 21. Histological results revealed that tissue architecture especially the epidermal integrity and dermal-epidermal junction of the Afp1m cryopreserved skin grafts exhibited better histological appearance, good preservation of tissue architecture and structural integrity than glycerolized skin. However, there was no significant difference among these groups in other histological criteria. There were no significant differences among the 4 groups in skin graft mechanical properties namely maximum load. In conclusion, Afp1m were found to be able to preserve the microstructure as well as the viability and function of the skin destined for skin transplantation when was kept at −4 °C for 72 h.     

Mechanical forces: The missing link between idiopathic pulmonary fibrosis and lung cancer

Patients with idiopathic pulmonary fibrosis (IPF) have a high risk of developing lung cancer compared with the general population. The morbidity of lung cancer in IPF patient ranges from 3% to 22%, and in some cases exceeds 50%, and these patients have a reduced survival time. However, the mechanisms through which IPF increases the morbidity and mortality in lung cancer remain unclear.By carefully analyzing the pathological features of these two diseases, we uncovered that, first, similar to IPF, lung carcinomas are more frequently found in the peripheral area of the lungs and, second, lung cancers tend to develop from the honeycomb areas in IPF. In accordance with the above pathological features, due to the spatial location, the peripheral areas of the lung experience a high stretch force because the average distance between adjacent alveolar cells in this area tends to be larger than that at the central lung when inflated; furthermore, the honeycomb areas, comprised of condensed fibrous tissue, are characterized by increased stiffness. Both of these pathological features of lung cancer and IPF are coincidentally related to abnormal mechanical forces (stretch and tissue stiffness). Therefore, we believe that the aberrant mechanical forces that are generated in the lung with IPF may contribute to the onset and progression of lung cancer.In this review, we discuss the possible effects of mechanical forces that are generated in IPF on the initiation and progression of lung cancer from the perspective of the hallmarks of cancer, including proliferation, metastasis, angiogenesis, cancer stem cells, immunology, epigenetics, and metabolism, so as to advance our understanding of the pathogenesis of IPF-related lung cancer and to harness these concepts for lung cancer mechanotherapies.     

Functional lung imaging with synchrotron radiation: Methods and preclinical applications

Many lung disease processes are characterized by structural and functional heterogeneity that is not directly appreciable with traditional physiological measurements. Experimental methods and lung function modeling to study regional lung function are crucial for better understanding of disease mechanisms and for targeting treatment. Synchrotron radiation offers useful properties to this end: coherence, utilized in phase-contrast imaging, and high flux and a wide energy spectrum which allow the selection of very narrow energy bands of radiation, thus allowing imaging at very specific energies. K-edge subtraction imaging (KES) has thus been developed at synchrotrons for both human and small animal imaging. The unique properties of synchrotron radiation extend X-ray computed tomography (CT) capabilities to quantitatively assess lung morphology, and also to map regional lung ventilation, perfusion, inflammation and biomechanical properties, with microscopic spatial resolution. Four-dimensional imaging, allows the investigation of the dynamics of regional lung functional parameters simultaneously with structural deformation of the lung as a function of time. This review summarizes synchrotron radiation imaging methods and overviews examples of its application in the study of disease mechanisms in preclinical animal models, as well as the potential for clinical translation both through the knowledge gained using these techniques and transfer of imaging technology to laboratory X-ray sources.     

Mechanics of root growth

Summary A model is developed for the rate of elongation of a root tip in terms of the balance of pressures acting on the root. Differentials of this equation give expressions for the changes in root elongation rate with respect to soil water potential and soil mechanical resistance. The model predicts that root cells osmoregulate against both water stress and soil mechanical resistance with predicts that root cells osmoregulate against both water stress and soil mechanical resistance with similar efficiencies which are less than 100%. Analysis of published data leads to the conclusion that root tips of pea osmoregulate with 70% efficiency. A working equation is developed for the elongation rate of roots in conditions of combined water stress and mechanical resistance.     

Physiological and biomechanical differences between wheelchair-dependent and able-bodied subjects during wheelchair ergometry

The purpose of this study was to compare the physiological and biomechanical responses of wheelchair-dependent persons (WCD) to able-bodied persons (AB) during manual wheelchair ergometry. Five WCD and five AB performed a discontinuous wheelchair ergometer test starting at 12.8 W at 30 rev.min-1 (57 m.min-1) with increments of 7.0 W at 6-min intervals. Biomechanical data were collected 3.5 min into each stage followed by the collection of physiological data. After the fifth stage, peak oxygen consumption was determined by having the subject work against a resistance of 14.7-19.6 N at 30 rev.min-1. The WCD had significantly higher net mechanical efficiency at 26.7, 33.6 and 40.6 W in comparison to the AB. The WCD had significantly greater shoulder extension at the point of initial wheel contact as measured by the shoulder angle, while the AB had significantly greater shoulder range of motion at all work rates in comparison to the WCD. The results demonstrate that a significant physiological difference exists in the manner by which WCD and AB accomplish wheelchair ergometry. The biomechanical differences between AB and WCD were found to be a prominent factor contributing to the higher mechanical efficiency of WCD over AB. It was concluded that basic physiological and biomechanical differences exist between WCD and AB in manual wheelchair locomotion and that these differences are important considerations to the interpretation of data in wheelchair ergometry studies.     

Constitutive and transport-related endocytotic pathways in turtle bladder epithelium

Summary Proton secretion in the urinary bladder of the fresh-water turtle is mediated by a proton pump located in the apical membrane of a population of cells characteristically rich in carbonic anhydrase. Earlier studies have demonstrated that these cells exhibit apical-membrane endocytotic and exocytotic processes which are thought to be involved in the regulation of the rate of proton transport via alterations in the number of pumps within the apical membrane. In this study, we sought to characterize these processes using two different methods. Analysis of transepithelial impedance yielded estimates of membrane capacitance which could be related to membrane area, thereby allowing one to monitor net changes in apical-membrane area resulting from changes in the net rates of endo-and exocytosis. Uptake of the fluid-phase marker FITC-dextran provided a measure of net extracellular volume uptake which was related to net rates of endocytosis. Our major conclusions are summarized as follows. The bladder cells exhibit a high baseline rate of endocytosis which appears to be a constitutive process similar to pinocytosis. This process is completely inhibited when ambient temperature is reduced to 15°C. In addition, serosal application of 0.5mm acetazolamide causes a transient increase in the rate of endocytosis, concomitant with a decrease in the rate of transport. Reduction of ambient temperature to 15°C reduces the rate of acetazolamide-induced endocytosis, but does not abolish it. Addition of 1mm serosal azide not only prevents the acetazolamide-induced increase in endocytosis, but also prevents the decrease in transport caused by acetazolamide. Azide has no effect on the baseline rate of endocytosis, nor does it prevent inhibition of carbonic anhydrase by acetazolamide. The specificity of azide, coupled with the different temperature sensitivities, demonstrate that the constitutive and transport-dependent endocytotic pathways are distinct processes. The observation that azide prevents both the acetazolamide-induced increase in endocytosis and the decrease in transport strongly supports the notion that endocytosis of proton-pump-containing membrane is requisite for the inhibition of transport by acetazolamide. Finally, the results also demonstrate that acetazolamide does not inhibit proton secretion simply by inhibiting carbonic anhydrase.     

Mechanical impedance increases aluminium tolerance of soybean (Glycine max) roots

The effect of aluminium (Al) on root elongation was studied in solution culture and sand culture. Compared to solution culture, in sand culture a ten times higher Al supply was necessary to inhibit root elongation to a comparable degree. This was due to a much lower Al uptake into the 5 mm root tips in sand culture. Fe concentrations in root tips were also lower in sand culture. Ca concentrations were higher and less depressed by Al, whereas Mg and K concentrations were not affected by the culture substrate. Regressions of Al concentrations in root tips versus inhibition of root elongation by Al revealed root damage at lower Al concentrations in sand culture. The effect of culture substrate on Al tolerance was independent of N source and could also be shown in flowing solution culture with and without sand. The results indicate that mechanical impedance in sand culture decreased Al uptake. This may be due to enhanced exudation of organic complexors thus reducing activites of monomeric Al species.     

Complete mechanical impedance increases the turgor of cells in the apex of pea roots

In this paper we describe an experimental approach which allows turgor (p) in an impeded root to be measured without the need to remove the root from the impeding environment. The maximum axial growth pressure (σ_max) generated by completely impeded pea (Pisum sativum L.) roots was measured using a novel apparatus incorporating a force transducer. The apparatus was designed so that it was possible to gain access to the impeded root with the microcapillary of a pressure probe and so obtain in situ measurements of P. Turgor in cells in the apical region of impeded roots was 0.78 MPa, compared with 0.55 MPa in unimpeded roots. In impeded roots, σ_max was 0.52 MPa, showing that the pressure component resulting from cell wall tension (W, where W=P–σ) decreased from 0.55 to 0.26 MPa as the roots became impeded. When impeded roots were removed from the apparatus, there was no decrease in P over the following 90 min. Impedance did not cause P to change in the non-elongating part of the roots further from the apex.     

An impedimetric method for rapid screening of cosmetic preservatives

An efficient impedance method was developed for rapid evaluation of cosmetic preservatives. The method used decimal reduction time or D-value to assess preservative efficacies. The D-value, which was calculated from the plot of Log CFU ml^–1 versus time by linear regression analysis, could be obtained within 48 h. Thus, the time required for the challenge test was reduced from 4–8 weeks with the standard procedures (eg US Pharmacopeia), to 2 days with the current method. A calibration curve (r=-0.95) was established by plotting the Log CFU ml^–1 versus capacitance detection time (DT) of 108 samples. With the calibration, CFU can be estimated directly from the impedance test without plating. Two commercial biocides and several other chemicals were evaluated in a shampoo by the impedance procedure againstPseudomonas aeruginosa. The D-values obtained from the impedance test were not significantly different from those produced by the conventional plate count method. The technique was found to be particularly useful when screening a large number of compounds to find novel preservatives and synergistic preservative combinations.