The potential for intercellular mechanical interaction: simulations of single chondrocyte versus anatomically based distribution

Computational studies of chondrocyte mechanics, and cell mechanics in general, have typically been performed using single cell models embedded in an extracellular matrix construct. The assumption of a single cell microstructural model may not capture intercellular interactions or accurately reflect the macroscale mechanics of cartilage when higher cell concentrations are considered, as may be the case in many instances. Hence, the goal of this study was to compare cell-level response of single and eleven cell biphasic finite element models, where the latter provided an anatomically based cellular distribution representative of the actual number of cells for a commonly used \(100 \, \upmu \hbox {m}\) edge cubic representative volume in the middle zone of cartilage. Single cell representations incorporated a centered single cell model and eleven location-corrected single cell models, the latter to delineate the role of cell placement in the representative volume element. A stress relaxation test at 10% compressive strain was adopted for all simulations. During transient response, volume- averaged chondrocyte mechanics demonstrated marked differences (up to 60% and typically greater than 10%) for the centered single versus the eleven cell models, yet steady-state loading was similar. Cell location played a marked role, due to inhomogeneity of the displacement and fluid pressure fields at the macroscopic scale. When the single cell representation was corrected for cell location, the transient response was consistent, while steady-state differences on the order of 1–4% were realized, which may be attributed to intercellular mechanical interactions. Anatomical representations of the superficial and deep zones, where cells reside in close proximity, may exhibit greater intercellular interactions, but these have yet to be explored.     

Dimensions and distribution of intercellular spaces in cryo-planed soybean nodules

The ability of legume nodules to regulate their permeability to gas diffusion has been attributed to physiological control over the size and distribution of gas-filed intercellular spaces within the nodule cortex. To examine the size and distribution of intercellular spaces and to determine whether they were filled with gas (high diffusion permeability) or liquid (low diffusion permeability), whole nodules were frozen in liquid nitrogen slush (-210°C), and then either cryo-fractured or cryo-planed before being examined by cold-stage scanning electron microscopy (SEM). The cryo-planed tissue was found to have many advantages over cryo-fractured nodules in providing images which were easier to interpret and quantify. Intercellular spaces throughout the nodule were examined in both tangential and medial planed faces. Since no differences were observed between views in either the size or shape of the open intercellular spaces, it was concluded that the intercellular spaces of nodules were not radially oriented as assumed in many mathematical models of gas diffusion. The inner cortex region in the nodules had the smallest intercellular spaces compared to other zones, and less than 10% of the intercellular spaces were occluded with any type of material in the central zone regions. Vacuum infiltration of nodules with salt solutions and subsequent cryo-planing for SEM examination showed that open and water-filled intercellular spaces could be differentiated. The potential is discussed for using this method to study the mechanism of diffusion barrier regulation in legume nodules.     

The distribution of hydrolytic enzyme activities in human fibroblast cultures and their intercellular transfer.

Both N-acetyl-β-D-glucosaminidase A and B (EC 3.2.1.30) are continuously secreted by normal cultured fibroblasts and can be taken up by deficient Sandhoff cells without cellular contact. The absence of intercellular transfer of β-galactosidase (EC 3.2.1.23) and acid α-glucosidase (EC 3.2.1.20) in cocultivations of normal and deficient fibroblasts is accompanied by very low extracellular activities of these enzymes in cultures of normal fibroblasts. For each of the hydrolases tested an appreciable amount of activity was found in the “pericellular” fraction. N-acetyl-β-D-glucosaminidase which has been taken up by deficient Sandhoff cells has an intracellular half-life of 6 days. The ingested intracellular enzyme, which is presumably localized in the lysosomes, is partly transferred to the pericellular fraction, and to the extracellular fraction. The results are discussed in relation to the secretion-recapture model proposed by Hickman and Neufeld.     

Frequency specificity in intercellular communication. Influence of patterns of periodic signaling on target cell responsiveness.

Cells often communicate by means of periodic signals, as exemplified by a large number of hormones and by the aggregation of Dictyostelium discoideum amebas in response to periodic pulses of cyclic AMP. Periodic signaling allows bypassing the phenomenon of desensitization brought about by constant stimuli. To gain further insight into the efficiency of pulsatile signaling, we analyze the effect of periodic stimulation on the dynamic behavior of a receptor system capable of desensitization toward its ligand. We first show that the receptor system adapts to square-wave stimuli, i.e., the response eventually reaches a steady, periodic pattern after a transient phase. By analyzing the dependence of the response on the characteristics of the square-wave stimulation, we show that there exist a waveform and a period of that signal that result in maximum responsiveness of the target system. Similar results are obtained when the signal takes the more realistic form of a periodically repeated stimulation followed by exponential decay of the ligand. The results are discussed with respect to the role of pulsatile secretion of gonadotropin-releasing hormone (GnRH) by the hypothalamus and of periodic signaling by cyclic AMP pulses in Dictyostelium. The analysis accounts for the existence, in both cases, of an optimal frequency and waveform of the periodic stimulus that correspond to maximum target cell responsiveness.     

Protein clefts in molecular recognition and function.

One of the primary factors determining how proteins interact with other molecules is the size of clefts in the protein's surface. In enzymes, for example, the active site is often characterized by a particularly large and deep cleft, while interactions between the molecules of a protein dimer tend to involve approximately planar surfaces. Here we present an analysis of how cleft volumes in proteins relate to their molecular interactions and functions. Three separate datasets are used, representing enzyme-ligand binding, protein-protein dimerization and antibody-antigen complexes. We find that, in single-chain enzymes, the ligand is bound in the largest cleft in over 83% of the proteins. Usually the largest cleft is considerably larger than the others, suggesting that size is a functional requirement. Thus, in many cases, the likely active sites of an enzyme can be identified using purely geometrical criteria alone. In other cases, where there is no predominantly large cleft, chemical interactions are required for pinpointing the correct location. In antibody-antigen interactions the antibody usually presents a large cleft for antigen binding. In contrast, protein-protein interactions in homodimers are characterized by approximately planar interfaces with several clefts involved. However, the largest cleft in each subunit still tends to be involved.     

The current distribution and potential for future range expansion of feral ferret Mustela putorius furo in Ireland

Predicting the potential invaded range of a non-native species is an important tool for conservation biology. The ferret (Mustela putorius furo) is a known invasive species outside its native range and has recently been confirmed as a feral species in Ireland. To determine the current distribution of feral populations, an all-island survey was conducted during 2006–2008. Using the results of this survey, a landscape modelling approach, using presence-only data was applied to predict the potential future range of this species in Ireland, given the availability of suitable habitat. The results suggest that Ireland appears to be potentially highly suitable for ferrets and, therefore, the possible ecological impacts of this species in Ireland are discussed.     

Effect of conditioning potential on potassium current kinetics in the frog node.

The kinetics of potassium conductance changes were determined in the voltage clamped frog node (Rana esculenta), as a function of conditioning prepotential. The conditioning potential duration varied from 1 to 50 ms and the amplitude between -60 and +130 mV (relative to rest). The conductance kinetics were determined at a single test potential of +20 mV (depolarization) by means of the slope of log [ninfinity - nt] vs. time relationship which defines the time constant of the process (tau). The values of tau, after conditioning hyperpolarizations, were around 5 ms, up to 10 times greater than values obtained following a strong depolarization. The tau vs. pre-potential curve was sigmoid in shape. These differences were only slightly dependent on [K+]0 or conditioning pulse duration. The steady-state current values were also found to be a function of conditioning potential. After conditioning hyperpolarizations, the log [ninfinity - nt] vs. time curve could not be fitted by a single exponent regardless of the power of n chosen. The prepotential dependency of potassium current kinetics is inconsistent with the Hodgkin-Huxley axon model where the conductance parameters are assumed to be in either one of two possible states, and where the rate of transfer from one state to the other follows first order kinetics. In contrast the described kinetics may be consistent with complex multistate potassium "channel" models or membranes consisting of a number of types of channels.     

Influence of phospholipid unsaturation on the cholesterol distribution in membranes.

Over the last half decade, we have studied saturated and unsaturated phosphatidylcholine (PC)-cholesterol membranes, with special attention paid to fluid-phase immiscibility in cis-unsaturated PC-cholesterol membranes. The investigations were carried out with fatty acid and sterol analogue spin labels for which reorientational diffusion of the nitroxide was measured using conventional ESR technique. We also used saturation recovery ESR technique where dual probes were utilized. Bimolecular collision rates between a membrane-soluble square-planar copper complex,3-ethoxy-2-oxobutyraldehyde bis(N4,N4-dimethylthiosemicarbazonato)copper(II) (CuKTMS2) and one of several nitroxide radical lipid-type spin labels were determined by measuring the nitroxide spin-lattice relaxation time (T1). The results obtained in all these studies can be explained if the following model is assumed: 1) at physiological temperatures, fluid-phase micro-immiscibility takes place in cis-unsaturated PC-cholesterol membranes, which induces cholesterol-rich domains in the membrane due to the steric nonconformability between the rigid fused-ring structure of cholesterol and the 30 degrees bend at the cis double bond of the alkyl chains of unsaturated PC. 2) The cholesterol-rich domains are small and/or of short lifetime (10(-9) s to less than 10(-7) s). Our results also suggest that the extra space that is available for conformational disorder and accommodation of small molecules is created in the central part of the bilayer by intercalation of cholesterol in cis-unsaturated PC membrane due to the mismatch in the hydrophobic length and nonconformability between cis-unsaturated PC alkyl chains and the bulky tetracyclic ring of cholesterol.     

Effect of sanguinarine, a benzophenanthridine alkaloid, on frog skin potential difference and short circuit current.

Sanguinarine, a benzophenanthridine alkaloid, causes a initial stimulation of frog skin short circuit current Isc when present in the mucosal bathing medium at 10(-4) M. The stimulation is accompanied by an increase in spontaneous potential difference (PD) and increase in D.C. resistance. No effects are seen with sanguinarine in the serosal bathing medium. The initial stimulation is followed by a decrease in Isc and PD, but a continued increase in resistance. In skins whose initial spontaneous PD is high, no initial stimulation in Isc and PD is seen; however, clamping these skins to a lower potential does not alter their initial inhibitory response to sanguinarine. Likewise, clamping the lower potential skins to higher potential does not alter their initial stimulatory response. Sanguinarine seems to be acting on the permeability barriers at the outer surface of the frog skin.     

Influence of draining on soil phosphorus forms and distribution in a constructed wetland

Seasonal water-table fluctuations in wetlands can result in flooded and drained conditions in the surface soil. In constructed wetlands water level drawdown and soil drainage are used in management to consolidate detrital materials, accelerate soil build up, and provide easy access for other management operations. A greenhouse study was conducted using intact peat soil cores to evaluate the changes in bioavailable P and other fractions following draining and reflooding. Measurements of floodwater dissolved reactive P (DRP) indicated that draining and soil exposure could result in large P flux to the overlying water column. Phosphorus flux in soils drained for 6 weeks was 10-fold higher (334 mg P m⁻² day⁻¹) than in soils drained for 3 weeks (33 mg P m⁻² day⁻¹). Soil exposure also resulted in an increase in bioavailable inorganic P (estimated by KCl extraction) at the expense of labile organic P pool. The KCl-P pool, which was initially less than 2% of total P (TP), increased to 3% and 13% of TP after 3 and 6 weeks draining, respectively. Results suggest that various soil P fractions, particularly those in newly accreted materials, were highly unstable and could be released in a more available form when newly accreted soils undergo drying. Water level drawdown and reflooding could result in significant P release, a possible stimulation of algal blooms and other water quality problems. Therefore, soil characteristics and chemistry and their impact on water quality should be a major consideration when one adopts the flood-drain technique in wetland management.     

Reduced current distribution of Psittacidae on the Mexican Pacific coast: potential impacts of habitat loss and capture for trade

Ecological niche models provide useful predictions of species distributions, but may fail to detect reductions in distribution due to factors other than habitat loss, such as hunting or trade. From 2001 to 2009, we conducted field-surveys along the Mexican Pacific coast to obtain presence–absence data for nine Psittacidae species. We applied Genetic Algorithm for Rule set Prediction (GARP) ecological niche modeling, using field-survey presence data to determine the potential current distribution of each species, and incorporated absence data to delineate extirpation areas. All parrot species showed a reduced current distribution, ranging from 9.6 to 79% reduction of estimated original distribution. The threatened and endemic species of Amazona oratrix, Amazona finschi, and Forpus cyanopygius suffered the greatest distribution reduction, higher than previously estimated by habitat-based models, suggesting that capture for trade may have caused extirpation of these species. The greatest extent of current distribution was occupied by Aratinga canicularis, Amazona albifrons and Ara militaris, which continue to occur throughout most of their original distribution. Amazona auropalliata, Aratinga strenua, and Brotogeris jugularis also occur throughout their restricted distribution in coastal Chiapas, and show a relatively small distribution reduction, but had the highest proportion of modified lands within their current distributions. Our results highlighted the regions of coastal Guerrero, northern Nayarit, and southern Sinaloa where parrot species have been extirpated even though GARP models predicted suitable habitat available. Ideally distribution models should be verified in the field to determine conservation priorities, and efforts should be directed to maintain populations of species with greatest distribution reductions.     

Blockers of potassium current and resting membrane potential in rat muscle fibers.

Rat diaphragm fibers were equilibrated for several hours in 150 mM KCl; when they were returned to 5 mM KCl the resting potential went back to its original level with a half time of 17 min. This repolarization was blocked by 5 mM BaCl2, a blocker of the inward rectifier K channel. On the other hand, 0.1 mM apamin and 0.02 mM glibenclamide which block the Ca-dependent and ATP sensitive K channels, respectively, and 0.1 mM 9-AC a blocker of the Cl- channel did not affect the repolarization. 5 mM barium decreased the K conductance measured under current-clamp conditions in diaphragm muscle fibers. The possible role of the inward rectifier system in the repolarization following return to normal [K]o is discussed.     

Voltage clamp simulations for multifiber bundles in a double sucrose gap: radial vs. longitudinal resistance effects

Voltage clamp responses of a single excitable fiber were simulated using a core conductor model including a high external resistance (Rs) in series to the fiber membrane to allow for intercellular clefts in a multifiber preparation. In terms of specific resistance, Rs was between 68 and 264 omega cm2. Internal resistivity (Ri) was taken to be zero or 200 omega cm. The aim of the study was to quantify the expected antagonistic effects of external and internal resistances on Na current measurements. With Ri = 0, the external resistance was found to cause a strong depression of fast inward current compared to an ideal space clamp at command potentials between -30 and 30 mV. With Ri = 200 omega cm, the depression of inward current was partially removed. The effects of Rs and Ri on membrane current measurement were illustrated by cable analysis assuming a quasi-steady state of the fiber at peak time of inward current.     

Influence of skin temperature distribution on thermal sensation in a cool environment

Thermal sensation and distribution of skin temperatures in persons exercising at 36.5 W on a bicycle ergometer and resting in a cool environment (10 degrees C) in two different clothings, one with the insulation mainly over the trunk (1.22 clo), and one with well insulated limbs (1.67 clo), were studied. Their general thermal sensations varied from slightly warm to slightly cool. The placing of the insulation had a decisive influence on skin temperature distribution, so that skin temperature was always high in well-insulated areas. When the insulation was placed over the limbs, a greater amount of heat was lost than if a similar insulation was placed on the trunk. Neither Tsk nor skin temperature distribution correlated with general thermal sensation. Instead, mean body temperature seemed to be the determinant of general thermal sensation in these conditions. The best prediction of general thermal sensation was obtained by adding Tre with a weighting factor of 0.8-0.9 and Tsk with a weighting factor of 0.1-0.2.