Control of sodium permeability of the outer barrier in toad skin

Summary The²⁴Na efflux (J _eff ^Na ) (i.e., the rate of appearance of²⁴Na in the outer compartment) in the isolated short-circuited toad skin bathed by NaCl-Ringer's solution on both sides is composed of para- and transcellular components of almost equal magnitudes. This relies on the assumption that amiloride acts on the transcellular component only and could block it completely.Ouabain induces a large transient increase of the transcellular component. This increase, which starts within a few minutes after the addition of ouabain, is due to electrical depolarization of the outer barrier, rather than a consequence of blocking Na recirculation across the inner barrier. The subsequent decline ofJ _eff ^Na , which takes place after the ouabain-inducedJ _eff ^Na peak, is due to a progressive block of outer barrier Na channels with time, which can eventually be complete, depending on the duration of action of ouabain. As the external Na concentration was always kept high and constant in these experiments, the results indicate that a rise in cell Na concentration, and not in the outer bathing solution, is the signal that triggers the reduction of outer barrier Na permeability (P ₀ ^Na ).Ouabain has no effect uponJ _eff ^Na with Na-free solution bathing the outer and NaCl-Ringer's solution the inner skin surface, showing the importance of Na penetration across the outer barrier, and not across the inner barrier due to its low Na permeability, in the process of closing the Na channels of this structure.Step changes from Na 115mm to Na-free external solution, or vice-versa, may affect both the outer barrier electrical potential difference (PD₀) and cell Na concentration (Na) _c . Therefore, the behavior ofJ _eff ^Na depends on which variable (if PD₀ or (Na) _c regulated outer barrier Na permeability) is most affected by step changes in outer bathing solution Na concentration.Amiloride in the control condition blocks the transcellular component ofJ _eff ^Na . However, in the condition of approximate short-circuiting of the outer barrier and high cellular Na concentration induced by long term effects of ouabain, when the Na channels of the outer barrier are already blocked by elevated cell Na concentration, amiloride may induce the opposite effect, increasing Na permeability of the outer barrier.With outer barrier Na channels completely blocked by high cell Na concentration, PCMB in the outer bathing medium induces a large increase ofJ _eff ^Na , rendering these channels again amiloride sensitive.The results are consistent with the notion that Na efflux from cell compartment to the outer bathing solution goes through the amiloride-sensitive Na channels of the apical border of the superficial cell layer of toad skin, with an apparent Na permeability modulated by cell ionic environment, most probably the cell Na concentration.The ensemble of the present results are consistent with Na permeability regulation taking place at the outer barrier level. However, this precise location could only be made unambiguously by measurements across the individual outer cell membranes.     

Identification of an uncommon haptoglobin type using DNA and protein analysis

The inherited variations in haptoglobin phenotypes are attributed to the homozygous and heterozygous combinations of three common autosomal alleles:HP ^* 1F,HP ^* 1S andHP ^* 2.HP ^* 1F andHP ^* 1S encode polypeptides that differ by two amino acids at positions 51 and 53. The formation ofHP ^* 2 is postulated to have resulted from a breakage and subsequent reunion event at non-homologous positions of twoHP ^* 1 alleles. The most common form ofHP ^* 2 isHP ^* 2FS in which the 5 end ofHP ^* 2 resemblesHP ^* 1F and the 3 end resemblesHP ^* 1S. Homologous crossing over betweenHP ^* 2 and either anHP ^* 1F orHP ^* 1S allele inHP ^* 2/HP ^* 1 heterozygotes can change the usual type ofHP ^* 2 to three other forms:HP ^* 2SS,HP ^* 2FF orHP ^* 2SF. We describe a nuclear family in which the uncommon genotypeHP ^* 2SS in one parent caused initial confusion in assigning genotypes to the rest of the nuclear family. The data demonstrate the need for a cautious approach when deducing haptoglobin genotypes from molecular analysis alone.     

A comparison between computer modeled bioaerosol dispersion and a bioaerosol field spray event

The observed deposition pattern from a field spray ofBacillus subtilus var.niger spores is compared with that of a computer simulated bioaerosol particle dispersion model. Using the same meteorological conditions as the field spray, the model produced a bioaerosol deposition pattern estimated to be reasonably similar (R ²=0.66) to the observed field pattern. Reasons for the differences between the deposition patterns are discussed. The comparison indicates that viable airborne particle deposition models may, with future testing, be useful tools for predicting near source aerial microbial dispersal and deposition. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.     

Enhanced Erythrocyte Adhesiveness/Aggregation in Obesity Corresponds to Low‐Grade Inflammation

Objective: Previous studies have suggested that obesity enhances the inflammatory response, producing macromolecules involved in the induction and/or maintenance of increased erythrocyte aggregation. The objectives of this study were to evaluate the correlation between inflammation markers, erythrocyte adhesiveness/aggregation, and the degree of obesity and to assess phosphatidylserine expression on erythrocyte surface membrane of obese vs. nonobese individuals. Research Methods and Procedures: Erythrocyte adhesiveness/aggregation in the peripheral venous blood was evaluated by using a new biomarker, phosphatidylserine expression was assessed by means of flow cytometry, and markers of inflammation were measured in 65 subjects: 30 obese [body mass index (BMI) = 41 ± 7.7 kg/m²] and 35 nonobese (BMI = 24 ± 2.7 kg/m²) individuals. Pearson correlations and Student's t test were performed. Results: A highly significant difference was noted in the degree of erythrocyte adhesiveness/aggregation and markers of inflammation between the study groups. BMI correlated with erythrocyte adhesiveness/aggregation (r = 0.42, p = 0.001), erythrocyte sedimentation rate (r = 0.42, p = 0.001), high‐sensitive C‐reactive protein (r = 0.55, p < 10^?4), fibrinogen (r = 0.37, p = 0.004), and white blood cell count (r = 0.45, p < 10^?4). The degree of erythrocyte adhesiveness/aggregation correlated with erythrocyte sedimentation rate (r = 0.5, p < 10^?4), high‐sensitive C‐reactive protein (r = 0.56, p < 10^?4), fibrinogen (r = 0.54, p < 10^?4), and white blood cell count (r = 0.32, p = 0.01). Discussion: Our results suggest that obesity‐related erythrocyte adhesiveness/aggregation is probably mediated through increased concentrations of adhesive macromolecules in the circulation and not necessarily through hyperlipidemia or phosphatidylserine exposure on erythrocyte's membrane.     

Introduction of a plant intron into the luciferase gene ofPhotinus pyralis

We describe a new luciferase reporter gene,luc ^INT, for early detection of luciferase activity inAgrobacterium transformation studies, and present improved techniques for the extraction of luciferase that decrease the time needed to quantitate luciferase activity. Theluc ^INT reporter gene combines the PIV2 intron fromGUS ^INT withluc ^*, the modified luciferase gene.luc ^INTis expressed in plant cells but not inAgrobacterium, allowing earlier detection of gene expression in the presence ofAgrobacterium during transformations in tobacco leaf discs. Stable expression levels ofluc ^INT andluc ^* in tobacco suspension cultures are compared for two different promoters. The nucleotide sequence data for the gene will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number U84006.     

Localization of mechanoreceptors in the protozoan,Stentor coeruleus

Summary Mechanoreceptor channels were localized by using the ligands, tubocurarine (TC), decamethonium (Deca), and gallamine (Gall), which have been shown to bind specifically to these channels. The binding of radioactively labeled TC (TC^*) was found to be directly proportional to the cell surface area suggesting that the channels are uniformly distributed over the cell surface. Intracellular TC and Gall injections did not depress mechanical stimulus sensitivity though these drugs did depress sensitivity when applied extracellularly at the same concentrations; therefore, the ligand binding sites are on or near the external surface of the cell. Autoradiographs revealed that radioactively labeled Deca (Deca^*) bound to the pigmented stripes but not to the ciliary stripes or membranellar band. Further,Stentor induced to shed their membranellar band through exposure to 8% urea were more sensitive to mechanical stimuli than were controls; therefore, the membranellar cilia do not appear to contain mechanoreceptor channels. Collectively, these data indicate that the mechanoreceptor channels are located in the somatic surface covering the pigmented stripes. The density of mechanoreceptor channels in the plasma membrane covering the somatic surface is tentatively estimated to be between 5500 and 14500 m^–2 based on the density of TC^* binding, the apparent number of TC molecules binding per mechanoreceptor channel, and data suggesting that only one fifth to one fourth of the bound TC^* is bound to structures in the plasma membrane.Abbreviations TC Tubocurarine - TC ^{* 14} C-tubocurarine - Deca Decamethonium - Deca ^{* 3}H-decamethonium - Gall Gallamine - DAPA Bis(3-aminopyridinium)-1,10-decane diiodide - DAPA ^{* 3}H-bis(3-ammopyridinium)-1,10-decane diiodide     

Possible reduction of surface charge by a mutation inParamecium tetraurelia

Summary Under voltage clamp, a mutant ofParamecium tetraurelia (teaB) shows a shift in the positive direction of the voltage sensitivity of the Ca conductance and the depolarization inactivation curve by 10 mV with no change in the total conductance. This effect can be mimicked in the wild type by the addition of external Ca²⁺ or Mg²⁺. The mutation also shifts the resting potential and the voltage sensitivities of the delayed rectification (depolarization-sensitive) K conductance and the anomalous rectification (hyperpolarization-sensitive) K conductance in the positive direction to a similar extent. This systematic shift of channel voltage sensitivities is best explained by the reduction of the surface negative charges of the membrane due to the mutation.     

Effects of membrane potential and surface potential on the kinetics of solute transport

A theoretical study has been made of the influence of the transmembrane potential difference and the surface potential of living cells on the kinetics of carried-mediated solute transport. It is assumed that the form of the free energy barrier within the membrane may be approximated by one dominant symmetrical peak, and that the electrical field is constant. Both single-ion transport kinetics and cotransport of an ion with a neutral solute are dealt with. Provided that the surface potential and the transmembrane potential are constant, the concentration dependence of the uptake rate is given by the Michaelis-Menten equation. The kinetic parameters, the maximal rate of uptake and the K_m, depend on both the surface potential and the membrane potential in a rather complex way. It is shown that the intuitive notion, that the maximal rate of cation uptake will increase when the cell membrane is hyperpolarized, is wrong in its generallity. Both an increase or a decrease may occur, depending on the characteristics of the transport system involved. If the magnitude of the membrane potential and the surface potential depends on the substrate concentration, marked deviations from Michaelis-Menten kinetics may come to the fore. This may result in either apparent positive or apparent negative homotrope cooperative effects. Enhancement of the uptake rate of the substrate ion may occur on adding another cation, despite the fact that the membrane will become depolarized. The same type of complex transport kinetics as found for Rb⁺ and Na⁺ uptake in yeast cells can be simulated by using a single-site transport model and including effects of the membrane potential.     

Thermodynamic aspects of plant cell adhesion to polymer surfaces

Summary A thermodynamic model of particle adhesion from a suspension onto a solid surface is used to predict the extent of adhesion of suspension-cultured Catharanthus roseus cells to the following polymer substrates: fluorinated ethylene-propylene (FEP), polystyrene (PS), polyethylene terephthalate (PET), sulphonated polystyrene (SPS), and glass. According to this model, the extent of adhesion is determined by the surface tensions of the plant cells, the polymer substrates, and the suspending liquid medium. Experimentally, adhesion of the washed plant cells was found to decrease with increasing substrate surface tension, following the sequence FEP>PS>PET>SPS>glass, when the surface tension of the liquid was greater than that of the plant cells, in agreement with the model. However, adhesion increased with increasing substrate surface tension when the liquid surface tension was lower than the cellular surface tension, also in agreement with the model. When the liquid and cellular tensions were equal the extent of adhesion was independent of the substrate surface tension. This also agrees with model predictions and leads to a value for the surface tension of C. roseus cells of approximately 54 ergs/cm² which is in agreement with a value obtained from contact angle measurements on layers of cells and sedimentation volume analysis. The cellular surface tension determined by the sedimentation volume method showed a biphasic alteration during growth cycles of C. roseus cell cultures. These variations (between 55 and 58 ergs/cm²) agree with the pattern of adhesion previously described.     

Purification and characterization ofFus sI3596, a 65 kd allergen ofFusarium solani

A component ofFusarium solani (F. solani), identified as the major allergen,Fus sI₃₅₉₆ ^* was purified to homogeneity from culture filtrate (CF) by means of anion-exchange column chromatography, gel filtration and FPLC. The homogeneity ofFus sI₃₅₉₆ ^* was assessed by IEF, PAGE, SDS-PAGE (non-reducing), immunoblot and HPLC.Fus sI₃₅₉₆ ^* was isolated as a glycoprotein of MW 65 kd and pI 3.6. The IgE ELISA-inhibition assay after periodate treatment of the fraction showed a lower IgE binding capacity suggesting involvement of carbohydrate moiety in IgE binding reactions of the allergen. Peptide fragments ofFus sI₃₅₉₆ ^* obtained after CNBr and trypsin treatment were analysed by immunoblotting for their allergenicity. This study indicated that there could be at least 3 allergenic determinants in the major allergen,Fus sI₃₅₉₆ ^* ofF. solani CF.Abbreviations DEAE diethyl aminoethyl - HPLC high performance liquid chromatography - SDS sodium dodecyl sulphate - PBS-Tween phosphate buffer saline containing 0.1% tween - DTT dithiothreitol - TFA triflouroacetate - FPLC fast protein liquid chromatography     

Kinetics of ionic transport across frog skin: Two concentration-dependent processes

Summary Sodium and chloride influxes across the nonshort-circuited isolated skin ofRana esculenta were measured at widely varying external ionic concentrations.The curve describing sodium transport has two Michaelis-Menten components linked at an inflection point occurring at an external sodium concentration of about 7 meq. Chloride transport can also be represented by two saturating components. A possible explanation of these kinetics is discussed.At sodium concentrations lower than 4 meq it is possible to define a component of the sodium transport mechanism as having a high affinity for sodium and which is independent of the nature of the external anion. A high affinity for chloride of the chloride transport system functioning at low external concentrations is also found but is significantly different from that of sodium. These systems show the physiological characteristics of the countertransports (Na _ext ⁺ /H _int ⁺ ; Cl _ext ^– /HCO _3int ^– ) functioning at low external concentrations.At external concentrations higher than 4 meq a low affinity transporting system in which chloride and sodium are linked superimpose on the high affinity components.The physiological significance of these results is discussed.     

Slow low-threshold potassium current inHelix pomatia neurons

A low-threshold outward current was studied in the neurons ofHelix pomatia at –70 to –30 mV using a two-electrode voltage clamp technique. In addition to the well-known A current (I _A), a slower outward current calledI _As (slow) was revealed. Activation and inactivation times ofI _As at –40 mV ranged from 90 to 120 msec and from 3 to 5 sec, respectively. The current recovered within 2 to 5 sec after inactivation at –120 mV. Analysis of changes in the reversal potential ofI _As caused by an increase in external potassium concentration suggests a potassium origin forI _As. The curves ofI _As stationary activation and inactivation fit the Boltzmann equation. Deriving from an activation curve, the activation potential for a half-maximum current,, is –40 mV, and the slope factor,k, is –9.8 mV, while those values for the inactivation curve are –84 mV (a half-maximum inactivation) and 7.5 mV.I _As is blocked by 4-aminopyridine (1–30 µM), tetraethylammonium (1 mM), and Ba²⁺ (1 mM), but is resistant to Cs⁺ (1 mM). PeakI _As is not affected either by substitution of external Ca²⁺ for Mg²⁺ or by application of Cd²⁺ (0.5–1.0 mM). The results suggest that activation ofI _As does not require Ca²⁺ entry into the cell.Neirofiziologiya/Neurophysiology, Vol. 25, No. 6, pp. 427–432, November–December, 1993.     

Reproduction rate,feeding process,and liebig limitations in cell populations—II

A biological system consisting of a population of cells suspended in a liquid substrate is considered. The general problem addressed in the paper is the derivation of the kinetic pattern of population growth as a statistical effect of a very large number of elementary interactions between a single cell and the molecules of nutrient in substrate. Solution of the problem is obtained in the form of equation expressing the population growth ratec as a function of substrate concentration,C _s. The analytical expression derived is applied to a real bacterial population (Escherichi coli) and kinetic patterns are theoretically computed. The major findings, expressed roughly, without nuances, are: (i) the concentration of nutrient at the cell membrane,C _c, can only be equal to either 0 (for theC _s below some threshold valueC ^*) orC _s (forC _s>C ^*); (ii) the Michaelis-Menten-Monod kinetics observed in experiments is an artifact: the pure (not contaminated by foreign factors) dependence ofc onC _s is actually such that the functionc=c(C _s) has practically linear increase whenC _s<C ^*, and is constant,c=c(C ^*)=const, whenC _s>C ^*; (iii) the Liebig principle is strictly fulfilled: up to a feasible accuracy of observation, under no circumstances can population growth be limited (controlled) by more than one substrate component—replacement of a limiting component for another one is an instant event rather than a gradual process.     

Morphological changes inEscherichia coli andBacillus cereus caused by external histone

It was found that the externally added histone changes remarkably both the surface and the internal ultrastructure of cells ofEscherichia coli. The interaction of histone with surface structures results in thickening of the inner layer of the cell wall. Cytoplasm becomes condensed, contains extensive electrontransparent zones and neither ribosomes nor the nuclear structure are differentiated. The addition of histone to germinating spores ofBacillus cereus decelerates germination and postgerminative development of this organism and changes ultrastructure of the external surface of the exosporium. The addition of Mg²⁺ ions reverting the effect of histone results in a renewal of the original ultrastruoture of the exosporium.     

Two distinct adhesion mechanisms in embryonic neural retina cells. I. A kinetic analysis

The reaggregation kinetics of embryonic chick neural retina cells prepared using several different dissociation procedures were monitored through decreases in the small-angle light scattering of aggregating samples. Two distinct modes of aggregation were revealed, one Ca²⁺ independent, the other Ca²⁺ dependent, suggesting the existence of two separate adhesion mechanisms. By varying the concentrations of Ca²⁺ and trypsin in the dissociation medium, we obtained cells which exhibited both, either, or neither mode of aggregation. The Ca²⁺-independent adhesiveness is active in the absence of proteolysis, is resistant to low levels of trypsin (0.001%), but is readily inactivated at higher trypsin concentrations in either the presence or absence of Ca²⁺. It is relatively temperature independent. By contrast, the Ca²⁺-dependent adhesiveness is not detected before exposure of the cells to proteolysis. It is expressed after tryptic proteolysis in the presence of Ca²⁺ and is then highly temperature dependent. It is resistant to further digestion by trypsin in the continued presence of Ca²⁺ but is lost when Ca²⁺ is subsequently removed, apparently through the expression of tryptic cleavage incurred earlier. We suggest that its increased activity may result at least in part from the clustering of surface components into adhesive patches. A provisional model is presented correlating these data.     

Effects of cytochalasin B and local anesthetics on electrical and morphological properties in L cells

Spontaneous oscillations of membrane potential observed in L cells were inhibited rapidly and reversibly in the presence of cytochalasin B (CB). Sustained hyperpolarization induced by high external Ca²⁺ was also depressed by the drug. However, Ca²⁺ injection into the cytoplasm elicited a sustained hyperpolarization, even in the presence of CB. These observations strongly suggest that CB inhibits calcium transport system in cell membrane. Morphological alterations associated with the CB treatment were decreased adhesiveness and rounding of the cells, with concomitant changes in surface architecture. Similar changes in electrophysiological and morphological properties were observed in cells treated with local anesthetics. Since such morphological changes induced by CB and local anesthetics were always preceded by electrical changes, it was suggested that the morphological changes are secondary phenomena resulting from inhibition of the Ca²⁺ transport.     

Microclimatic differences between and within canopy gaps in a temperate rainforest

The microclimate in the understorey and in two canopy gaps of different size (249 m² and 12.6 m²) within a temperate rainforest in northern New Zealand were characterised using net all-wave radiation (Q ^*), vapour pressure deficit (VPD), vertical air temperature gradient (VTG), and maximum and minimum air temperatures. Hemispherical fisheye photographs were used to define the exposure to the sky at each microclimate recording site both within and between gaps, and in the understorey. Under condiions of clear skies during the summer, with the sun close to its maximum altitude, the large gap centre had a mean daytime (0700–1700 hours)Q ^* of 452 W/m². This value was similar to that estimated for an open field nearby, but three times greater thanQ ^* at the centre of the small gap, and six times greater than in the understorey. The east and west sides of both gaps had higher values ofQ ^* than the north and south sides. BothQ ^* and mean daily maximum temperature were strongly correlated with the amount of sky exposure. VPD and VTG were higher in gaps than in the understorey, with maximum values for both variables occurring at the centre of the large gap.     

Manganese as a calcium probe: Electron paramagnetic resonance and nuclear magnetic resonance spectroscopy of intact cells

Summary When Lettree cells are exposed to Mn²⁺, the cation becomes associated with cells in two ways: in a relatively loose and mobile manner that gives a six-line EPR spectrum designated Mn _b ^*, and in an immobile, relatively tight manner that gives no detectable EPR spectrum, designated Mn _b . Mn _b ^* is probably on the surface of cells; most Mn _b is probably inside cells. NMR measurements of Lettree cell suspensions show two water proton relaxation rates and confirm the existence of cell-associated Mn. Human erythrocytes, on the other hand, bind no Mn²⁺ under these conditions, as judged by EPR and NMR measurements.Virally-treated Lettree cells show an increase in Mn _b (but not in Mn _b ^*). They also show a third water proton relaxation rate.     

Short-term sedimentation in Tagus estuary,Portugal: the influence of salt marsh plants

Short-term sediment deposition was studied at four salt marsh areas in the Tagus estuary. In areas covered with Sarcocornia perennis, Sarcocornia fruticosa, Halimione portulacoides and Spartina maritima and also in the non-vegetated areas, sedimentation was measured as the monthly accumulation of sediments on nylon filters anchored on the soil surface, from August 2000 to May 2001. Our experiments were used also to determine the influence of the different plant species in vertical accretion rates. Short-term sedimentation rates (from 2.8 to 272.3 g m⁻² d⁻¹) did show significant differences when the four salt marshes studied in the Tagus estuary were compared to each others. Salt marshes closer to the sediment sources had higher sedimentation rates. Our results suggest that the salt marsh type and surface cover may provide small-scale variations in sedimentation and also that sediment deposition values do change according to the position of the different plant species within the salt marsh. Sedimentation is an essential factor in salt marsh vertical accretion studies and our investigation may provide support to help forecast the adaptative response of the Tagus estuary wetlands to future sea level rise.     

Periodic electric field as a biopolymer conformation switch: a possible mechanism

A theoretical model is proposed to describe the influence of a periodic electric field (PEF) upon a biopolymer. The biopolymer is treated as a classical mechanical system consisting of subsystems (molecular groups) which interact with each other through potential forces. The PEF is treated as a periodic driving force applied to a molecular group. The energy dissipation is considered using the model of fluid (viscous) friction. Arguments for the non-linear character of the friction-velocity dependence caused by the non-Newtonian rheology of a viscous medium are formulated.A forced molecular-group motion is investigated for the situation of a small driving-force period, with oscillations overdamped and a driving force consisting of more than one harmonic. As a result, it is established that the motion always gets to a terminal stage when only a small-scale vibration about some point, X ^*, takes place. The terminal motion is preceded by a transient characterized by the presence of a directional velocity component and so by a drift along a potential profile. the drift goes on until a barrier is met which has a sufficiently large steepness (the barrier height is not important). As a result, the point X ^* may happen to be remote from the conformation potential local minimum (conformational state). The physical reasons for the drift are described.If we consider the small-scale vibration about X ^* in the framework of the hierarchy of scales for intramolecular mobility, it can be regarded as an equilibrium mobility, whereas the drift can be regarded as a functionally important motion, and X ^* as a new conformational state which is realizable only in the presence of the PEF. It may be concluded, as the result of a consistent treatment and neglecting the small-scale vibration, that the conformational potential is modified by adding a linear term (in the one-dimensional case). In this connection, the set of conformational states can both deform (deviation of the positions of minima and their relative depth) and rearrange qualitatively (some minima can vanish and/or new minima can appear). In particular, the transition from one conformation to another one may happen due to rearrangement.