Mass-Action Expressions of Ion Exchange Applied to Ca, H, K, and Mg Sorption on Isolated Cells Walls of Leaves from Brassica oleracea

The cation exchange properties of cell walls isolated from collard (Bassica oleracea var acephala D.C.) leaves were investigated. Cation sorption on cell walls was described by mass-action expressions of ion exchange, rather than by the traditional Donnan equilibrium. The mass-action expressions enable the selectivity of the wall for one cation over another to be determined unambiguously from ion exchange isotherms. We found that: (a) the cation composition of the wall varied as a function of the solution cation concentration, solution cation composition, and pH in a way predicted by mass action; (b) the affinity of the wall for divalent cations increased as the equivalent fraction of divalent cation on the wall increased, and as the concentration of divalent cations in solution increased; (c) the selectivity of the wall for any metal cation pair was not altered by the concentration of H⁺ in solution or on the wall; (d) H⁺ sorption on the wall may be treated as a cation exchange reaction making it possible to calculate the relative affinity of the wall for metal cation pairs from H⁺-metal (Me) titration curves; and (e) the relative affinity of the wall for the cations we studied was: H⁺ (K⁺ ≥ Ca²⁺) > Mg²⁺. A cation-exchange model including surface complexes is consistent with observed cation selectivity. We conclude that metal cations interact with the wall to minimize or eliminate long-range electrostatic interactions and suggest that this may be due to the formation of site-specific cation-wall surface complexes.     

Chemical basis for selectivity of metal ions by the Bacillus subtilis cell wall.

The use of equilibrium dialysis techniques established that isolated cell walls of Bacillus subtilis possess selective affinities for several cations. The binding of these cations to the cell wall was influenced by the presence of various functional groups in the peptidoglycan matrix. Selective chemical modification of the free carboxyl and amino groups showed that when amino groups were replaced by neutral, bulky, or negatively charged groups, the sites available for cation complexing generally increased. Introduction of positive charges into the wall resulted in a marked decrease in the numbers of metal binding sites and usually a decrease in the apparent association constants. Both teichoic acid and peptidoglycan contribute to the sites available for interaction with metals. Hill plots of equilibrium dialysis data suggest that metal binding to cell walls involves negative cooperativity. Competition between various metals for binding sites suggested that the cations complex with identical sites on the cell walls. When the hydrogen ion concentration was increased, the affinity of the walls for metals decreased, but the numbers of metal binding sites remained constant, suggesting that cations and protons also compete for the same sites.     

Capsule walls as barriers to oxygen availability: Implications for the development of brooded embryos by the estuarine gastropod Crepipatella dilatata (Calyptraeidae)

Encapsulation of developing embryos imposes potential restrictions, because the capsule wall must allow for adequate inward diffusion of oxygen and for increased diffusion of oxygen as metabolic demand increases with continued development. Samples of egg capsules from the gastropod Crepipatella dilatata were used to document surface characteristics, composition of the different capsule wall layers, and alterations in wall thickness during development. The diffusion coefficient and capsule wall permeability were determined experimentally for capsules containing embryos at different developmental stages. We also determined oxygen consumption rates for various embryonic stages and for nurse eggs, which provide food for embryos during development. The capsule wall of C. dilatata possesses 2 differentiated layers: the external capsular wall (ECW) and the internal capsular wall (ICW). The ECW is compact and fibrous, features that remain invariable during development, and lacks surface features that might make some portions of the capsule wall more permeable to oxygen than others. On the other hand, the ICW is initially spongy and thick, but significantly decreases in thickness over time, particularly before the embryos begin feeding on nurse eggs. Although the capsule wall is a serious barrier to diffusion, permeability to oxygen increases over time by 112% due to the dramatic thinning of the inner capsule wall layer. Nurse eggs consume oxygen but at very low rates, supporting the idea that they correspond to living embryonic cells that have stopped their development. Respiration measurements indicated that embryos are initially supplied with enough oxygen within the egg capsules to carry out the activities characteristic of embryogenesis, even though the capsular walls show their maximum thickness and lowest permeability at this time. However, as the embryo develops its velum and becomes more active, capsule wall thickness decreases and capsule permeability to oxygen increases. Correspondingly, the oxygen demands of metamorphosed but still encapsulated specimens are approximately 135% higher than those of pre-metamorphosed sibling embryos.     

The intertidal marine lichen formed by the pyrenomycete fungus Verrucaria tavaresiae (Ascomycotina) and the brown alga Petroderma maculiforme (Phaeophyceae): thallus organization and symbiont interaction

The thallus formed by the marine pyrenomycete fungus Verrucaria tavaresiae and the phaeophycean alga Petroderma maculiforme was studied to elucidate the organization of the symbionts, determine the type of cellular contacts between them, and evaluate the status of the symbiosis as a lichen. Hand-sectioned and resin-embedded samples were examined with light and transmission electron microscopy. Within the uppermost portion of the cellular fungal tissue, separate algal filaments were arranged anticlinally. Protrusions of the fungal cell wall penetrated into adjacent algal walls but did not enter the cell lumen. A striking feature of these penetrations was the frequent separation of algal cell wall layers and insertion of fungal wall material between them. Algal filaments grew downward intrusively between fungal cells, often penetrating deeply into the fungal cell wall. Despite the exceptional nature of the phycobiont involved, the Verrucaria tavaresiae-Petroderma maculiforme symbiosis unequivocally fits the prevailing concept of a lichen. The distinctive interpenetrations observed between symbionts may be related to the integration of their different growth forms within a coherent tissue regularly subject to mechanical stresses. Periclinal cell divisions within and just below the algal layer may serve to replenish surface tissues lost to abrasion and herbivory.     

Cell wall extensibility during branch formation in the xanthophycean alga Vaucheria terrestris

In the tip-growing filamentous cell of the xanthophycean alga Vaucheria terrestris sensu Götz, a new growing tip develops in the non-growing, cylindrical region of the cell that was exposed by local illumination. The present study examined changes in the strength and extensibility of the cell wall of the new growing tip and in the matrix components of the inner surface of the cell wall. The internal pressure required to rupture the cell walls decreased remarkably during the early to middle stages of growing tip development, but the cell wall hardly extended before rupture. In contrast, during the middle and late stages of development, cell walls were extended by internal pressure. Atomic force microscopy revealed that protease-resistant, fine granular matrix components were present only at the apical portion of a normal growing tip, and were absent in the non-growing cylindrical region. In the early and middle stages of new growing tip development, these matrix components appeared in the cell walls in patches. These results suggest that first cell wall strength decreases and then cell wall extensibility increases in the development of new growing tips, and that protease-resistant, fine granular matrix components may be involved in rendering a cell wall extensible.     

Water transport and ion-water interaction in the gramicidin channel

The diffuse permeability and the diffusion coefficient of water (Dw) in the gramicidin channel is determined from the osmotic water permeability of the channel and "single file" pore theory. Dw is about 7% of the self-diffusion coefficient of bulk water. The diffusion coefficient of a single water molecule alone in the channel is also determined and is about equal to the value in bulk water. This provides an estimate of the mobility of water on the channel walls in the absence of water-water interaction. Since the gramicidin channel walls should be representative of uncharged polar protein surfaces, this result provides direct evidence that the presence of a cation in the channel reduces the hydraulic water permeability by a factor ranging from 60 for Tl+ to 5 for Na+. The diffusion coefficient of a cation (Dc) in the channel is estimated and compared with Dw. For Na+ it is found that Dc approximately equal to Dw, which implies that the movement of the row of water molecules through the channel determines the local mobility of Na+. Thus, it seems that short range ion-wall interactions are not important in determining the channel conductance for Na+. In contrast, for Li+, local ion-wall interactions probably do limit the conductance.     

Exchange diffusion of alkali ions through the apoplast of the potato (Soianum tuberosum L.) storage parenchyma

Interdiffusion coefficients of K and Na for tangential transport through cell walls were found to be independent of ion strength and only moderately (c. 50%) lower than values known for diffusion in water. Although at low salt concentrations alkali ion exchange diffusion on the cell wall pathway is facilitated by accumulation of diffusing species in the Donnan space, at all concentrations the resistance of the whole tissue for apoplastic cation exchange diffusion remained significantly higher than that of unstirred liquid of equal thickness, as the area fraction of the cell wall pathway in the parenchyma is small.     

Effect of albumin on the structure of the molecular filter at the capillary wall

The removal of plasma proteins from a vascular perfusate results in increased labeling of the endothelial cell (EC) vesicles and increased permeability of the capillary wall to water and solutes. The hypothesis that albumin forms part of a molecular filter composed of a network of fibrous molecules is evaluated. The fibrous network covers the EC surface and penetrates the intercellular junctions. Albumin may simply occupy space within the matrix to increase the resistance to water flow and increase exclusion and restriction to diffusion of solutes. Electrostatic interactions between positively charged sites on albumin and negatively charged fibers may also order the fibrous network into a more selective array. In the presence of albumin, the fibrous network would determine the selectivity of the capillary wall. An alternative hypothesis, that a selective pathway is formed when albumin is adsorbed to the walls of the wide portion of the slit, is inconsistent with the area required for the diffusion of small solutes between the endothelial cells. However, the geometry of intercellular channels may partially determine the selectivity of the capillary wall when the fiber matrix containing albumin is disrupted.     

Staining and osmotic properties of young gametophytes ofPolypodium vulgare L. and their bearing on rhizoid function

Summary The rhizoids of gametophytes ofPolypodium vulgare L. rapidly absorb vital stains whereas the protonemal cells are impermeable to these stains, which can only enter the cells from the rhizoids. The protonemal cells which bear rhizoids were found to have a slightly higher osmotic equivalent than did the rhizoids or the protonemal cells on either side. From the results of several staining procedures it was demonstrated that the rhizoid walls contain free carboxyl groups and thus possess cation exchange properties. Most of the carboxyl groups are probably present in a yellow-brown wall matrix substance, which shows high resistance to acid and alkali extraction. The precise nature of this substance has not been determined but it could be an acid mucopolysaccharide. Carboxyl groups are detectable in the protonemal cell walls only after saponification and are probably esterified in the untreated wall. Several other chemical and physiological differences were found between the rhizoids and the protonemal cells and it was concluded that the specific properties of the rhizoids are related to their function as organs of uptake.     

Kinetics of release of serotonin from isolated secretory granules. II. Ion exchange determines the diffusivity of serotonin.

We measured the efflux of 5-hydroxytryptamine (5-HT, serotonin) from an intact secretory granule extracted from the mast cell of the beige mouse. The efflux was measured with amperometry after rupture of the granule membrane was triggered by electroporation. We determined the diffusivity of 5-HT within the secretory granule to be 2.0 x 10(-8) cm2 s(-1) when the granule is in contact with a physiological saline and found that this diffusivity depends on the valence of the cation in the external electrolyte. There is a fivefold increase in the diffusion coefficient of 5-HT determined in CsCl (150 mM, pH 7.2) at 3.7 x 10(-8) cm2 s(-1) compared to that determined in histamine dihydrochloride (Hi, 100 mM at pH 4.5) at 0.7 x 10(-8) cm2 s(-1). We found that the rate of expansion of the granule matrix observed in physiological medium correlates with the efflux of 5-HT, and that the rate of swelling of the matrix and the efflux depend on the microviscosity within the granule matrix and not the bulk viscosity of the external solution. The low diffusivity of 5-HT (approximately 500-fold less than in the bulk), the observation that the valence of the counterion affects this diffusivity, and the relationship between the volume changes of the matrix and the efflux suggest that 5-HT is released from the granule by ion exchange. We discuss the implications of this result for exocytotic release in mast cells and propose that an ion exchange mechanism could control the rate of release in other secretory systems.     

Characterization of the Neurospora crassa DHN melanin biosynthetic pathway in developing ascospores and peridium cells

Neurospora crassa contains all four enzymes for the synthesis of DHN (dihydroxynaphthalene), the substrate for melanin formation. We show that the DHN melanin pathway functions during N. crassa female development to generate melanized peridium and ascospore cell walls. N. crassa contains one polyketide synthase (PER-1), two polyketide hydrolases (PKH-1 and PKH-2), two THN (tetrahydroxynaphthalene) reductases (PKR-1 and PKR-2), and one scytalone dehydratase (SCY-1). We show that the PER-1, PKH-1, PKR-1 and SCY-1 are required for ascospoer melanization. We also identified the laccase that functions in the conversion of DHN into melanin via a free radical oxidative polymerization reaction, and have named the gene lacm-1 (laccase for melanin formation-1). In maturing perithecia, we show that LACM-1 is localized to the peridium cell wall space while the DHN pathway enzymes are localized to intracellular vesicles. We present a model for melanin formation in which melanin is formed within the cell wall space and the cell wall structure is similar to “reinforced concrete” with the cell wall glucan, chitin, and glycoproteins encased within the melanin polymer. This arrangement provides for a very strong and resilient cell wall and protects the glucan/chitin/glycoprotein matrix from digestion from enzymes and damage from free radicals.     

Differential interferometry in the analytical ultracentrifuge. II. General applications

Various ways of applying differential interferometry to ultracentrifugal analyses are examined and several analytical techniques are established. In transport and moving boundary methods, the sedimentation coefficient is more precisely determined in the differential interference system than in the schlieren optical system because fringe measurement accuracy is much higher in the former system. Compared to interference and absorption optics, the differential interferometer provides a more exact s value in the transport method since an accurate calculation procedure can be adopted. Moreover, the following advantages of differential interferometry are noted. Determination of the initial solute concentration, which must be done in the usual interference method, is unnecessary in this sedimentation equilibrium method. Regardless of the partial loss of solute from the observed system due to rapid precipitation or adsorption to the cell wall during centrifugation, the molecular weight of the rest of the solute can be determined exactly. The diffusion coefficient can be determined accurately by fringe displacement analysis at the hinge point during the transient state. Together with the molecular weight and diffusion coefficient, the partial specific volume and sedimentation coefficient of a solute can be obtained from the result of a single low-speed centrifugation when the sample solutions in H₂O and D₂O are compared.     

Micro-heterogeneity of pectins and calcium distribution in the epidermal and cortical parenchyma cell walls of flax hypocotyl

Summary Pectic polysaccharides are major components of the plant cell wall matrix and are known to perform many important functions for the plant. In the course of our studies on the putative role of pectic polysaccharides in the control of cell elongation, we have examined the distribution of polygalacturonans in the epidermal and cortical parenchyma cell walls of flax seedling hypocotyls. Pectic components have been detected with (1) the nickel (Ni²⁺) staining method to visualize polygalacturonates, (2) monoclonal antibodies specific to low (JIM5) and highly methylesterified (JIM7) pectins and (3) a combination of subtractive treatment and PATAg (periodic acid-thiocarbohydrazide-silver proteinate) staining. In parallel, calcium (Ca²⁺) distribution has been imaged using SIMS microscopy (secondary ion mass spectrometry) on cryo-prepared samples and TEM (transmission electron microscopy) after precipitation of calcium with potassium pyroantimonate. Our results show that, at the tissular level, polygalacturonans are mainly located in the epidermal cell walls, as revealed by the Ni²⁺ staining and immunofluorescence microscopy with JIM5 and JIM7 antibodies. In parallel, Ca²⁺ distribution points to a higher content of this cation in the epidermal walls compared to cortical parenchyma walls. At the ultrastructural level, immunogold labeling with JIM5 and JIM7 antibodies shows a differential distribution of pectic polysaccharides within cell walls of both tissues. The acidic polygalacturonans (recognized by JIM5) held through calcium bridges are mainly found in the outer part of the external wall of epidermal cells. In contrast, the labeling of methylesterified pectins with JIM7 is slightly higher in the inner part than in the outer part of the wall. In the cortical parenchyma cells, acidic pectins are restricted to the cell junctions and the wall areas in contact with the air-spaces, whereas methylesterified pectins are evenly distributed all over the wall. In addition, the pyroantimonate precipitation method reveals a clear difference in the Ca²⁺ distribution in the epidermal wall, suggesting that this cation is more tightly bound to acidic pectins in the outer part than in the inner part of that wall. Our findings show that the distribution of pectic polysaccharides and the nature of their linkages differ not only between tissues, but also within a single wall of a given cell in flax hypocotyls. The differential distribution of pectins and Ca²⁺ in the external epidermal wall suggests a specific control of the demethylation of pectins and a central role for Ca²⁺ in this regulation.Abbreviations Cdta diamino-1,2-cyclohexane tetra-acetic acid - PATAg periodic acid-thiocarbohydrazide-silver proteinate - PGA polygalacturonic acid - PME pectin methylesterase - RG I rhamnogalacturonan I - SIMS secondary ion mass spectrometry - TEM transmission electron microscopy     

Penetrability of White Rot-Degraded Pine Wood by the Lignin Peroxidase of Phanerochaete chrysosporium

The penetration of enzymes into wood cell walls during white rot decay is an open question. A postembedding immunoelectron microscopic technique was the method of choice to answer that question. Infiltration of pine wood specimens with a concentrated culture filtrate greatly improved the labeling density and, thereby, reproducibility. Characterization of the concentrated culture filtrate by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting (immunoblotting) revealed three closely spaced proteins of molecular weights about 42,000 showing immunoreactivity against anti-lignin peroxidase serum. It was shown by immunogold labeling that lignin peroxidase of Phanerochaete chrysosporium is located on the surface of the wood cell wall or within areas of heavy attack. It did not diffuse into undecayed parts of the cell wall. The reasons for preventing lignin peroxidase from penetrating wood cell walls during white rot decay are discussed.     

Effects of glucanase treatment on the structure and mechanical properties of cell walls in the giant‐celled xanthophycean alga Vaucheria frigida

The cell wall of the tip‐growing cells of the giant‐cellular xanthophycean alga Vaucheria frigida is mainly composed of cellulose microfibrils (CMFs) arranged in random directions and the major matrix component into which the CMFs are embedded throughout the cell. The mechanical properties of a cell‐wall fragment isolated from the tip‐growing region, which was inflated by artificially applied pressure, were measured after enzymatic removal of the matrix component by using a protease; the results showed that the matrix component is involved in the maintenance of cell wall strength. Since glucose and uronic acid are present in the matrix component of Vaucheria cell walls, we measured the mechanical properties of the cell wall after treatment with endo‐1,3‐ß‐glucanase and observed the fine structures of its surfaces by atomic force microscopy. The major matrix component was partially removed from the cell wall by glucanase, and the enzyme treatment significantly weakened the cell wall strength without affecting the pH dependence of cell wall extensibility. The enzymatic removal of the major matrix component by using a protease released polysaccharide containing glucose and glucuronic acid. This suggests that the major matrix component of the algal cell walls contains both proteins (or polypeptides) and polysaccharides consisting of glucose and glucuronic acid as the main constituents.     

Nitric oxide diffusion rate is reduced in the aortic wall

Endogenous nitric oxide (NO) plays important physiological roles in the body. As a small diatomic molecule, NO has been assumed to freely diffuse in tissues with a diffusion rate similar to that in water. However, this assumption has not been tested experimentally. In this study, a modified Clark-type NO electrode attached with a customized aorta holder was used to directly measure the flux of NO diffusion across the aortic wall at 37°C. Experiments were carefully designed for accurate measurements of the apparent NO diffusion coefficient D and the partition coefficient α in the aortic wall. A mathematical model was presented for analyzing experimental data. It was determined that α = 1.15 ± 0.11 and D = 848 ± 45 μm²/s (n = 12). The NO diffusion coefficient in the aortic wall is nearly fourfold smaller than the reported diffusion coefficient in solution at 37°C, indicating that NO diffusion in the vascular wall is no longer free, but markedly dependent on the environment in the tissue where these NO molecules are. These results imply that the NO diffusion rate in the vascular wall may be upregulated and downregulated by certain physiological and/or pathophysiological processes affecting the composition of tissues.     

The preprophase band: possible involvement in the formation of the cell wall.

Numerous vesicles were observed among the microtubules of the "preprophase" band in prophase cells from root tips of Allium cepa. The content of these vesicles looks similar to the matrix of adjacent cell walls, and these vesicles often appear to be involved in exocytosis. In addition, the cell walls perpendicular to the plane of (beneath) the preprophase band are often differentially thickened compared to the walls lying parallel to the plane of the band. Our interpretation of these observations is that the preprophase band may direct or channel vesicles containing precursors of the cell wall to localized regions of wall synthesis. The incorporation of constituents of the cell wall into a narrow region defined by the position of the preprophase band may be a mechanism that ensures unidirecitonal growth of meristematic cells.     

The distribution of cell wall polymers during antheridium development and spermatogenesis in the Charophycean green alga, Chara corallina

Background and Aims

The production of multicellular gametangia in green plants represents an early evolutionary development that is found today in all land plants and advanced clades of the Charophycean green algae. The processing of cell walls is an integral part of this morphogenesis yet very little is known about cell wall dynamics in early-divergent green plants such as the Charophycean green algae. This study represents a comprehensive analysis of antheridium development and spermatogenesis in the green alga, Chara corallina.

Methods

Microarrays of cell wall components and immunocytochemical methods were employed in order to analyse cell wall macromolecules during antheridium development.

Key Results

Cellulose and pectic homogalacturonan epitopes were detected throughout all cell types of the developing antheridium including the unique cell wall protuberances of the shield cells and the cell walls of sperm cell initials. Arabinogalactan protein epitopes were distributed only in the epidermal shield cell layers and anti-xyloglucan antibody binding was only observed in the capitulum region that initially yields the sperm filaments. During the terminal stage of sperm development, no cell wall polymers recognized by the probes employed were found on the scale-covered sperm cells.

Conclusions

Antheridium development in C. corallina is a rapid event that includes the production of cell walls that contain polymers similar to those found in land plants. While pectic and cellulosic epitopes are ubiquitous in the antheridium, the distribution of arabinogalactan protein and xyloglucan epitopes is restricted to specific zones. Spermatogenesis also includes a major switch in the production of extracellular matrix macromolecules from cell walls to scales, the latter being a primitive extracellular matrix characteristic of green plants.     

Electron Microscopy of the Microbial Populations Present and Their Modes of Attack on Various Cellulosic Substrates Undergoing Digestion in the Sheep Rumen

Cotton fibers and various cell wall preparations from grass leaves and from the feces of sheep fed on dried grass were placed in the sheep rumen in bags made from 5-μm-mesh nylon cloth. After periods of from 3 to 48 h, bags were removed, and the contents were fixed, embedded, sectioned, and stained for electron microscopy. Some of the bacteria present were seen to be closely associated with the cell walls, either tunneling within them or making very close contact. Evidence was obtained for differential digestion of cell walls and of the layers within them. Distinct differences were noticed between bacterial populations attacking the more susceptible wall types and those attacking feces cell walls and cotton fibers. Among the latter, the dominant form was a long, thin rod with a typical gramnegative cell wall structure, different from that described for Bacteroides succinogenes S85 or for Butyrivibrio fibrisolvens.