Effects of aluminum on plasma membrane as revealed by analysis of alkaline band formation in internodal cells of Chara corallina.

To study the mechanism of aluminum toxicity in plant cells, the effects of aluminum on alkaline band formation were analyzed in the internodal cells of Chara. After cells were treated with AlCl3, they were examined for their capacity to develop alkaline bands. Treating cells with AlCl3 medium at pH 4.5 completely inhibited alkaline band formation. When either CaCl2 or malic acid was added to the AlCl3 medium (pH 4.5), it did not produce an ameliorative effect, whereas addition of both CaCl2 and malic acid induced a significant ameliorative effect. It was found that treatment at pH 4.5 in the absence of AlCl3 strongly inhibited alkaline band formation. This inhibition by the low pH (4.5) treatment was effectively ameliorated by CaCl2. At higher pH (5.0), malic acid alone produced a significant ameliorative effect on aluminum inhibition of alkaline band formation, but CaCl2 did not. Recovery from aluminum inhibition was also studied. When cells treated with AlCl3 at pH 4.5 were incubated in artificial pond water, they could not recover the capacity to develop alkaline band. When either malic acid or CaCl2 was added to artificial pond water, cells recovered their alkaline band formation. It was concluded that one of the primary targets of aluminum is the plasma membrane and that aluminum affects the plasma membrane from the cell exterior at the beginning of the treatment (within 24 h). It was also suggested that the aluminum treatment impairs the HCO3- influx mechanism but not the OH- efflux mechanism.     

PH-dependent cell–cell interactions in the green alga Chara

Protoplasma - Characean internodal cells develop alternating patterns of acid and alkaline zones along their surface in order to facilitate uptake of carbon required for photosynthesis. In this...     

Structural Characteristics of Developing Nitella Internodal Cell Walls

The Nilella intermodal cell is formed by a division of the segment cell, the latter being a direct derivative of the shoot apical cell. The internodal cell is remarkable in that it elongates from an initial length of about 20 microns to a mature length of about 60 millimeters. The structures of the apical and segment cells, and the internodal cells in all stages of development were examined with the techniques of interference, polarization, and electron microscopy. The apical and segment cells were found to be isotropic. The upper part of the segment cell, destined to form a node, shows a curious pitted structure that was characteristic of certain node structures. The lower part of the segment cell, destined to become an internodal cell, shows a vague transverse arrangement of fibrils at the inner wall surface. The internodal cells, from the time they are first formed, show negative birefringence and a transverse arrangement of microfibrils at the inner wall surface. The elongation of the internodal cell is characterized by a rise, dip, and rise in both the optical thickness and retardation of the cell wall. The dip in both these variables coincides with the attainment of the maximum relative elongation rate. After the cessation of elongation, wall deposition continues, but the fibrils at .the inner surface of the wall are now seen to occur in fields of nearly parallel microfibrils. These fields, with varying fibrillar directions, may partly overlap each other or may merge with one another. Unlike the growing wall, this wall which is deposited after the end of elongation is isotropic.     

Cyclosis-related asymmetry of chloroplast–plasma membrane interactions at the margins of illuminated area in <Emphasis Type="Italic">Chara corallina</Emphasis> cells

Cytoplasmic streaming in plant cells is an effective means of intracellular transport. The cycling of ions and metabolites between the cytosol and chloroplasts in illuminated cell regions may alter the cytoplasm composition, while directional flow of this modified cytoplasm may affect the plasma membrane and chloroplast activities in cell regions residing downstream of the illumination area. The impact of local illumination is predicted to be asymmetric because the cell regions located downstream and upstream in the cytoplasmic flow with respect to illumination area would be exposed to flowing cytoplasm whose solute composition was influenced by photosynthetic or dark metabolism. This hypothesis was checked by measuring H⁺-transporting activity of plasmalemma and chlorophyll fluorescence of chloroplasts in shaded regions of Chara corallina internodal cells near opposite borders of illuminated region (white light, beam width 2 mm). Both the apoplastic pH and chlorophyll fluorescence, recorded in shade regions at equal distances from illuminated area, exhibited asymmetric light-on responses depending on orientation of cytoplasmic streaming at the light–shade boundary. In the region where the cytoplasm flowed from illuminated area to the measurement area, the alkaline zone (a zone with high plasma membrane conductance) was formed within 4-min illumination, whereas no alkaline zone was observed in the area where cytoplasm approached the boundary from darkened regions. The results emphasize significance of cyclosis in lateral distribution of a functionally active intermediate capable of affecting the membrane transport across the plasmalemma, the functional activity of chloroplasts, and pattern formation in the plant cell.     

Fluorescence and Photosynthetic Activity of Chloroplasts in Acid and Alkaline Zones of Chara corallina

Continuous profiles of local pH near the cell surface of Chara corallinawere recorded during uniform longitudinal movement of an internodal cell relative to a stationary pH microelectrode. Under illumination, the pH profile consisted of alternating acid and alkaline bands with a pH difference of up to 3 pH units. After darkening, the bands disappeared and pH became uniformly distributed along the cell length. Chlorophyll fluorescence of chloroplasts was measured by microfluorometry at different locations within one cell, and significant differences were observed in close relation to light-dependent pH banding. The chlorophyll fluorescence yield was lower in zones of low external pH than in alkaline zones both under actinic and saturating light. The fluorescence parameters Fand F" _m and the quantum yield of photosystem II (PSII) displayed variations along the cell length in accordance with pH changes in unstirred layers of the medium. The results show that PSII photochemical efficiency and the rate of noncyclic electron transport are higher in the chloroplasts of acid zones (zones of H⁺extrusion from the cell) than in alkaline zones. The dependence of photosynthetic electron transport on local pH near the cell surface may result from different contents of CO₂in acid and alkaline regions. The acid zones are enriched with CO₂that readily permeates through the membrane providing the substrate for the Calvin cycle. Conversely, a poorly permeating form, HCO^– ₃is predominant in alkaline zones, which may restrict the dark reactions and photosynthetic electron flow.     

THE ASSOCIATION OF ROSETTE AND GLOBULE TERMINAL COMPLEXES WITH CELLULOSE MICROFIBRIL ASSEMBLY IN NITELLA TRANSLUCENS VAR. AXILLARIS (CHAROPHYCEAE)1,2

A freeze-fracture investigation of the putative cellulose synthesizing complex (terminal complex) morphology in Nitella translucens var. axillaris (A. Br.) R.D.W. internodal cells revealed single solitary EF globules and PF rosettes on the plasma membrane. The average density of rosettes in elongating internodal cells was 5.6 μm^?2 with slight spatial variation observed. In only three other algal genera (all zygnematalean) have rosette / globule terminal complexes been observed, while this characteristic is common to all vascular plants and one moss thus far investigated. This evidence strongly suggests that the rosette type of terminal complex morphology is an additional characteristic of charophycean algae and lends further support to the hypothesis that this group of algae represents the evolutionary line that gave rise to vascular plants. Observations were also made from the freeze-fracture of Nitella internodal cells concerning the orientation of cell wall microfibrils and cytoskeletal elements near the plasma membrane. The pattern of microfibril orientation in growing internodal cells is initially transverse to the cell long axis, becoming progressively axial presumably due to the strain of elongation. In mature internodal cells, the pattern of microfibril orientation is helicoidal. Microtubules appressed to the inner surface of the plasma membrane are oriented parallel to the most recently formed microfibrils in elongating and mature internodal cells.     

Periodic band pattern as a dissipative structure in ion transport systems with cylindrical shape

A theory is presented for appearance of periodic band patterns of ion concentration and electric potential associated with electric current surrounding a unicellular or multicellular system of a cylindrical shape. A flux continuity at the membrane (or the surface) is reduced to a nonlinear equation expressing passive and active fluxes across the membrane and intracellular diffusion flux. It is shown that, when an external parameter is varied from the sub-critical region, i.e. the homogeneous flux state, a symmetry breaking along a longitudinal axis usually appears prior to the one along a circumferential direction. The spectrum analysis shows that the correlation length is longer in the longitudinal direction. Growth of the band pattern from a patch-shaped pattern is demonstrated by the use of numerical calculations of proton concentration on the two-dimensional space of cylindrical surface. An experimental example of formative process of H⁺ banding is given for the internodal cell ofChara. It is shown that small patches on the surface decline or are sometimes gathered to the band surrounding the circle. The resulting pattern is suggested as a kind of dissipative structure appearing far from equilibrium.     

Optical activity of octopus metarhodopsins.

The optical activity of octopus rhodopsin, acid metarhodopsin and alkaline metarhodopsin was studied by a sensitive and rapid CD apparatus. For sometime it has been thought that cephalopod metarhodopsins do not have any optical activity associated with their main absorption band. However, the present work shows that acid metarhodopsin in digitonin has a positive CD band at 498 nm and a negative CD band at 436 nm and alkaline metarhodopsin has a negative CD band at 381 nm. Detergent affected the wavelength of the CD peak of the visual pigments though the pattern of the spectrum was similar. From these results it is concluded that the conformation of all-trans retinal in octopus metarhodopsin is influenced by the asymmetric conformation of the protein near the retinal and therefore inducing optical activity.     

Intracellular perfusion and cell centrifugation studies on plasmalemma transport processes inChara corallina

Summary The OH^– transport system ofChara corallina was studied using the techniques of intracellular perfusion and cell centrifugation. Application of silk ligatures to internodal cells had quite a perturbative effect on the OH^– transport activity. Approximately 12 hr were required before normal pH profiles were reestablished.Tonoplast-free internodal cells developed rather weak, uniform alkalinity along the cell surface. Redevelopment of control pH patterns was never observed in these experiments. Surface pH profiles similar to those observed on tonoplast-free cells could also be obtained by subjecting cells to mild centrifugation (180×g). Organelles of the streaming cytoplasm were contained within the centrifugal cell segment by applying a ligature near the cell center. Normal pH profiles were observed along the centrifugal segment, while the centripetal segment developed weak, rather uniform alkaline profiles. Upon redistribution of the cytoplasmic organelles, normal pH profiles were established along the entire cell length.These results indicate that an organelle within the streaming cytoplasmic phase is responsible for the spatial location and control over OH^– transport. This explains the absence of control pH profiles in tonoplast-free cells, since during the disintegration of the tonoplast, most of the streaming cytoplasm coagulates at one end of the cell.Parallel pH mapping and electrophysiological studies indicated that the plasmalemma of this species contains an ATP-dependent electrogenic H⁺ transport system. Also, experiments conducted in the presence and absence of cellular ATP demonstrated that OH^– efflux can be driven passively by the membrane potential. Whether OH^– transport is strictly a passive process in normal cells remains to be resolved.     

Actin in living and fixed characean internodal cells: identification of a cortical array of fine actin strands and chloroplast actin rings

Summary We report on the novel features of the actin cytoskeleton and its development in characean internodal cells. Images obtained by confocal laser scanning microscopy after microinjection of living cells with fluorescent derivatives of F-actin-specific phallotoxins, and by modified immunofluorescence methods using fixed cells, were mutually confirmatory at all stages of internodal cell growth. The microinjection method allowed capture of 3-dimensional images of high quality even though photobleaching and apparent loss of the probes through degradation and uptake into the vacuole made it difficult to record phallotoxin-labelled actin over long periods of time. When injected at appropriate concentrations, phallotoxins affected neither the rate of cytoplasmic streaming nor the long-term viability of cells. Recently formed internodal cells have relatively disorganized actin bundles that become oriented in the subcortical cytoplasm approximately parallel to the newly established long axis and traverse the cell through transvacuolar strands. In older cells with central vacuoles not traversed by cytoplasmic strands, subcortical bundles are organized in parallel groups that associate closely with stationary chloroplasts, now in files. The parallel arrangement and continuity of actin bundles is maintained where they pass round nodal regions of the cell, even in the absence of chloroplast files. This study reports on two novel structural features of the characean internodal actin cytoskeleton: a distinct array of actin strands near the plasma membrane that is oriented transversely during cell growth and rings of actin around the chloroplasts bordering the neutral line, the zone that separates opposing flows of endoplasm.     

Influence of light on the apoplastic ph in microwounded cells of <Emphasis Type="Italic">Chara corallina</Emphasis>

Microscopic wounding of plant cell walls by pathogens or by feeding insects triggers the defense responses, including a sharp rise in pH at the cell surface (pH_o). Using internodal cells of Chara corallina Klein ex Willd., we show here that the elevated pH_o in the area of cell wall microincision decreases in darkness and increases on illumination. These pH_o changes occurred specifically in cell areas affected by microincision and were lacking in intact areas with active pHotosynthesis (acid zones). Localized illumination of a remote cell region located upstream the cytoplasmic flow at a 1.5-mm distance from the analyzed area also caused a transient increase in pH_o in the area of microwounding but had no such effect in unwounded cell regions having weakly acidic pH_o. Apparently, the increase in pH_o after wounding is mediated by a metabolite released from illuminated chloroplasts, which is transported with the cytoplasmic flow for long distances. The transient pH_o increase in the area of cell wall incision after illumination of a distant cell region coincided with a temporal increase in chlorophyll fluorescence F’. This implies the concurrent influence of the transported reductant (presumably NADH) on light emission of chloroplasts and on the H⁺ flow across the plasmalemma. We suppose that the alkalinization of cell surface in the area of microincision arises from H⁺ consumption in the apoplast in association with the transmembrane electron transport from cytoplasmic reducing equivalents to molecular oxygen.     

Cross-linking of a polyomavirus attachment protein to its mouse kidney cell receptor.

We used photoaffinity cross-linking with the heterobifunctional cross-linker N-hydroxysuccinimidyl 4-azidobenzoate (HSAB) to covalently link polyomavirus to a mouse kidney cell surface component. The virus-HSAB combination was adsorbed to the cells and then cross-linked and isolated in monopinocytotic vesicles from the cells after endocytosis. The cross-linked product was identified on sodium dodecyl sulfate-polyacrylamide gels by the presence of a new band carrying 125I-labeled virion protein with a higher molecular mass than the normal virion protein bands. A single new band, with an apparent molecular mass of 120 kilodaltons (120 kDa), was identified by this procedure. This band was formed only in the presence of the HSAB cross-linker when virions were bound to the cells. The band also copurified with cross-linked virions when virion-containing vesicles were treated with detergent to remove the cell membrane. Antibody treatments that blocked up to 100% of virus binding and internalization also blocked cross-linking, as measured by the formation of the 120-kDa band. The 120-kDa band was characterized by preparation of antibody against the excised band from the gel. This antibody was shown to have the expected dual specificity for polyomavirus VP1 sequences and plasma membrane proteins, as analyzed on Western blots. The anti-120-kDa antibody was also shown by immunofluorescence to bind to the surface of mouse kidney cells. These data have demonstrated that molecules of possible biological significance in the binding of polyomavirus to mouse kidney cells have been cross-linked and that cell surface molecules have been identified that may be characterized further for possible receptor function in polyomavirus attachment.     

Prolein polymorphism in a randombred chicken population

Polymorphism in plasma amylase, plasma alkaline phosphatase, non-specific esterase and red cell esterase-D of the Athens-Canadian randombred (ACRB) population of chickens was determined by polyacrylamide and starch gel electrophoresis. Amylase alleles Amy-1^A and Amy-1^B were segregating in the ACRB population with frequencies of 0.45 and 0.55 respectively. For the plasma alkaline phosphatase the F and S bands, the B band and a new isozyme migrating at a faster rate than the previously reported F band were detected. A genetic nomenclature for plasma alkaline phosphatase is suggested which considers the difference between the F and S bands as the presence or absence of sialic acid attached to a primary protein.
Plasma esterase activity was observed in all four of the regions previously reported, but there was no polymorphism found in any of the loci. All birds in this population showed the same red-cell esterase-D phenotype which consisted of a main band with sub-bands on each side.     

Expansion of the tetragonally arrayed cell wall protein layer during growth of Bacillus sphaericus.

The outermost layer of the cell wall of Bacillus sphaericus strain P-1 is a tetragonally arrayed structure (T-layer) which is assembled from a single polypeptide. No turnover of T-layer was detected during growth of cultures. In contrast, the turnover of peptidoglycan was between 20 and 25% per generation. The sites of deposition of new T-layer on the cell surface were identified by the indirect fluorescent antibody technique, which labeled old T-layer, and by the reverse technique, which labeled new T-layer. These experiments demonstrated that the major area of T-layer deposition was a band at the site of an incipient cell division. This band subsequently split and covered the new pole of each progeny cell. Little or no T-layer was inserted into existing poles. In addition, multiple bands of new T-layer, which probably accommodate cell elongation, were inserted along the lateral surface of the cell.     

Mercury-arc photolysis: a method for examining second messenger regulation of endothelial cell monolayer integrity.

Cell-cell apposition in bovine pulmonary endothelial cell monolayers was modulated by inducing transient increases in intracellular adenosine 3':5'-cyclic monophosphate (cAMP) and 1,4,5-inositol triphosphate (IP3). This was accomplished by mercury-arc flash photolysis of o-nitrobenzyl derivatives of the second messengers (caged compounds). Second messenger release by the mercury-arc lamp was determined by radioimmunoassay of cAMP to have a t1/2 of approximately 8 min. Each second messenger induced the phosphorylation of a distinct subset of cytoskeletal proteins; however, both IP3 and cAMP increased vimentin phosphorylation. Actin isoform patterns were not altered by the second messengers. Intracellular pulses of IP3 in pulmonary endothelial cells caused disruption of endothelial monolayer integrity as determined by phase-contrast microscopy and by visualization of actin stress fibers with rhodamine-phalloidin. Intracellular pulses of cAMP increased cell-cell contact, cell surface area, and apposition. IP3 appeared to have its greatest effect on the actin peripheral band. In silicone rubber contractility assays this agent caused contraction of pulmonary microvascular endothelial cells as visualized by an increase in wrinkles beneath the cells. On the other hand, cAMP appeared to effect both the peripheral band and centralized actin domains. Caged cAMP caused relaxation of endothelial cells as visualized by a disappearance of wrinkles beneath the cells.     

Effect of Glutaraldehyde on the Outer Layers of Escherichia coli

S ummary : Sodium lauryl sulphate (SLS) at pH 3 and 8 lysed cell walls of Escherichia coli. Pretreatment with glutaraldehyde at pH 3 and at pH 8 prevented this lysis. SLS induced maximum lysis of E. coli cells at 40°; pretreatment of cells with glutaraldehyde prevented this lysis also. Electrophoretic studies indicated that glutaraldehyde accumu lated on the surface of E. coli cells more rapidly in acid than in alkaline conditions, and that it blocked amino groups on the surface layer of Bacillus subtilis spores. The relationship of these findings to the bactericidal efficiency of glutaraldehyde in acid and alkaline solution is discussed.     

ELECTRON MICROSCOPE STUDIES OF THE FORMATION OF NODES OF RANVIER IN MOUSE SCIATIC NERVES

Observations with the electron microscope of longitudinal sections of the sciatic nerves of infant mice during the period of early myelin formation are described. These observations are interpreted in relation to previous studies of transverse sections, and a general picture of the formation of an internodal length of the myelin sheath in three dimensions is formulated. In general, an internodal length of myelin sheath is attained by the spiral wrapping of the infolded Schwann cell surface; the increase in length of the internode during maturation is at least partially explained by the increased length of axon covered by the overlapping of successive layers during the wrapping of the infolded Schwann cell surface; and the nodes of Ranvier refer to the structure complex at the junctions of adjacent non-syncytial Schwann cells. The fact that the mode of formation of myelin brings each of its layers into intimate contact with the axon surface at the nodes is emphasized because of the possible functional significance of this arrangement. The manner of origin of Schmidt-Lantermann clefts remains obscure. Certain isolated observations provide evidence for the possibility that occasional internodes of myelin may form from several small segments of myelin within a single Schwann cell.     

A culture substratum appropriate for brain cells is a chondroitin sulfate glycosaminoglycan in serum

When cells dissociated from the neonatal rat brains are plated on a poly-lysine-coated surface in a serum-free medium, they display a strange morphology: a dark and extended cell body. Preincubation of the surface with fetal bovine serum was found to inhibit the appearance of this strange contraction of the basal cell sheets in a dose-dependent manner. This finding indicated the presence of a factor(s) in the serum, which might be an appropriate substratum for prolonged survival of brain neurons. In the current study, this factor was highly purified through DEAE ion-exchange chromatography followed by gel filtration. The factor was eluted from a Superose column at fractions corresponding to a molecular weight greater than 1000 kDa. By SDS-PAGE analysis, these fractions were found to contain a major band (≥1000 kDa) positive for alcian blue and few minor bands faintly stainable with Coomassie blue. The activity of the purified sample, inducing the morphological change in cells, was diminished by incubation with chondroitinase ABC. Neither heparitinase II, hyaluronidase, nor trypsin modified the activity. An authentic chondroitin sulfate (type B) mimicked the serum action on the morphology of brain cells in early stages of culture. Taking these findings together, it is suggested that the factor in serum beneficial for the attachment of brain cells is composed of a chondroitin sulfate with a Mr greater than 1000 kDa. Cortical cells dissociated from the neonatal rat brain attached well to the purified factor-coated surface and displayed a healthy morphology: an optically-reflective cell body with thick neurites for at least 3 days in the absence of serum.     

Inversion of extracellular current and axial voltage profile inChara andNitella

Summary Reducing the pH of the bathing solution from 8.2 to pH 6 can induce an inversion of the extracellular current pattern that develops at the surface ofChara corallina internodal cells. A similar result can be obtained on some cells by changing the medium to a pH value of 10. In noninvertingChara cells the currents were strongly reduced when the pH value of the medium was changed between 3 and 11. Simultaneous measurements of theChara transmembrane potential in the acid and alkaline regions revealed that a light-induced electrical potential gradient of approximately 24 mV was present in the axial (or longitudinal) direction. Correlated to the external current pattern inversion was an inversion of this internal longitudinal voltage gradient. Reillumination ofNitella cells, after a period of darkness, often resulted in a complete inversion of the extracellular current pattern. These results are discussed in terms of spatial and temporal control of membrane transport processes, and in particular the control of current loops that pass through these cells.