Espin cytoskeletal proteins in the sensory cells of rodent taste buds

Espins are multifunctional actin-bundling proteins that are highly enriched in the microvilli of certain chemosensory and mechanosensory cells, where they are believed to regulate the integrity and/or dimensions of the parallel-actin-bundle cytoskeletal scaffold. We have determined that, in rats and mice, affinity purified espin antibody intensely labels the lingual and palatal taste buds of the oral cavity and taste buds in the pharyngo-laryngeal region. Intense immunolabeling was observed in the apical, microvillar region of taste buds, while the level of cytoplasmic labeling in taste bud cells was considerably lower. Taste buds contain tightly packed collections of sensory cells (light, or type II plus type III) and supporting cells (dark, or type I), which can be distinguished by microscopic features and cell type-specific markers. On the basis of results obtained using an antigen-retrieval method in conjunction with double immunofluorescence for espin and sensory taste cell-specific markers, we propose that espins are expressed predominantly in the sensory cells of taste buds. In confocal images of rat circumvallate taste buds, we counted 21.5 ± 0.3 espin-positive cells/taste bud, in agreement with a previous report showing 20.7 ± 1.3 light cells/taste bud when counted at the ultrastructural level. The espin antibody labeled spindle-shaped cells with round nuclei and showed 100% colocalization with cell-specific markers recognizing all type II [inositol 1,4,5-trisphosphate receptor type III (IP₃R₃)_, α-gustducin, protein-specific gene product 9.5 (PGP9.5)] and a subpopulation of type III (IP₃R₃, PGP9.5) taste cells. On average, 72%, 50%, and 32% of the espin-positive taste cells were labeled with antibodies to IP₃R₃, α-gustducin, and PGP9.5, respectively. Upon sectional analysis, the taste buds of rat circumvallate papillae commonly revealed a multi-tiered, espin-positive apical cytoskeletal apparatus. One espin-positive zone, a collection of ～3 μm-long microvilli occupying the taste pore, was separated by an espin-depleted zone from a second espin-positive zone situated lower within the taste pit. This latter zone included espin-positive rod-like structures that occasionally extended basally to a depth of 10–12 μm into the cytoplasm of taste cells. We propose that the espin-positive zone in the taste pit coincides with actin bundles in association with the microvilli of type II taste cells, whereas the espin-positive microvilli in the taste pore are the single microvilli of type III taste cells.     

A method for isolating adult and neonatal airway smooth muscle cells and measuring shortening velocity

Methods are described for isolating smooth muscle cells from thetracheae of adult and neonatal sheep and measuring the single-cell shortening velocity. Isolated cells were elongated,Ca²⁺ tolerant, and contractedrapidly and substantially when exposed to cholinergic agonists, KCl,serotonin, or caffeine. Adult cells were longer and widerthan preterm cells. Mean cell length in 1.6 mMCaCl₂ was 194 ± 57 (SD) µm(n = 66) for adult cells and 93 ± 32 µm (n = 20) for preterm cells(P < 0.05). Mean cell width at thewidest point of the adult cells was 8.2 ± 1.8 µm(n = 66) and 5.2 ± 1.5 µm(n = 20) for preterm cells(P < 0.05). Cells were loaded into aperfusion dish maintained at 35°C and exposed to agonists, andcontractions were videotaped. Cell lengths were measured from 30 videoframes and plotted as a function of time. Nonlinear fitting of celllength to an exponential model gave shortening velocities faster thanmost of those reported for airway smooth muscle tissues. For a sampleof 10 adult and 10 preterm cells stimulated with 100 µM carbachol,mean (± SD) shortening velocity of the preterm cells was notdifferent from that of the adult cells (0.64 ± 0.30 vs. 0.54 ± 0.27 s¹, respectively), butpreterm cells shortened more than adult cells (68 ± 12 vs. 55 ± 11% of starting length, respectively;P < 0.05). The preparative andanalytic methods described here are widely applicable to other smoothmuscles and will allow contraction to be studied quantitatively at thesingle-cell level.

     

Localisation of actin, villin, fimbrin, ezrin and ankyrin in rat taste receptor cells

Mammalian taste buds consist of 50–150 pear- or spindle-shaped taste receptor cells which contain, at their apical cell surface, a bundle of microvillar projections. The microvilli probably serve to increase the receptive membrane surface of the chemosensory receptor cells. The molecular basis controlling the ultrastructure of taste receptor microvilli is present unknown. In the present study we analysed, by immunostaining at the light and electron microscopic levels and by immunoblotting, components of the cytoskeleton of these microvilli. We show here that taste cell microvilli contain the major cytoskeletal proteins of intestinal microvilli, actin, fimbrin and villin. Another actin-binding, peripheral membrane protein of intestinal microvilli, ezrin, was also localised to taste cell microvilli, where ezrin might play a role, for example, in placement of specific membrane proteins to the microvillus membrane. In search of further linkage proteins, we found ankyrin localised along the basolateral cell surface of taste receptor cells, where ankyrin might be involved in the immobilisation of the Na⁺, K⁺-ATPase or other ion-translocating proteins of taste cells to the membrane cytoskeleton. Accepted: 26 April 1999     

Taste Bud Cell Generation in the Perihatching Chick

Chick taste bud primordia initially appear in late gestationon embryonic day 17 (E17), 4 days before hatching. To trackDNA synthesis and subsequent taste bud cell proliferation betweenE17 and the second day post-hatching (H2), single 25 µCiinjections of tritiated thymidine (specific activity = 72.5Ci/mmol) were administered in ovo during E15, E16, E17 or E18.Anterior mandibular oral epithelium was processed for lightmicroscopic autoradiography. Sections through each taste bud'scenter were analysed for label (6 silver grains/gemmal cellnucleus), and bud diameter. Results indicated a major part ofgemmal cell DNA synthesis does not occur until after E19 irrespectiveof the day of thymidine injection, suggesting postmitotic orquiescent (decycled) cells assemble to form the early bud primordium(E17–19) based on local tissue interactions. All budsexamined from E20–H2 contained labelled cells. The dayof injection was important since 5-day survival cases afterE16 injection yielded about 25% the number of labelled cells/budas compared with equivalent survival cases following E17–18injections. These results are discussed with respect to parallelchanges in bud shape and increasing bud diameter, and cell proliferationin possible extra- and intragemmal sources of bud cells.     

Distribution and morphology of the ampullary organs of the estuarine long-tailed catfish, <Emphasis Type="Italic">Euristhmus lepturus</Emphasis> (Plotosidae,Siluriformes)

Ampullary organs of Euristhmus lepturus occur in high densities along the head and in four parallel pathways along the trunk of the body. Large ampullary pores (125–130 μm) are easily distinguishable from other sensory epithelial pores due to the differences in size and the presence of a collar-like structure. Simple, singular ampullary organs of the head region consist of an ampullary pore connected to a long canal with a diameter of 115–175 μm before terminating as a simple ampulla with an external diameter of 390–480 μm. The ampullary canal is composed of 1–2 layers of flattened squamous epithelial cells, the basement membrane and an interlocking collagen sheath. The innermost cells lining the canal wall are adjoined via tight junctions and numerous desmosomes, as are those of the receptor and supportive cells. Canal wall tissue gives rise to a sensory epithelium containing between 242 and 285 total receptor cells, with an average diameter of 11.7 ± 5.3 μm, intermixed with medially nucleated supportive cells. Each receptor cell (21.38 ± 4.41 μm, height) has an apically positioned nucleus and a luminal surface covered with numerous microvilli. Neural terminals abut the basal region of receptor cells opposite multiple presynaptic bodies and dense mitochondria. Supportive cells extend from the ampullary lumen to the basement membrane, which is adjacent to the complex system of collagen fibres.     

PeakForce Tapping resolves individual microvilli on living cells

Microvilli are a common structure found on epithelial cells that increase the apical surface thus enhancing the transmembrane transport capacity and also serve as one of the cell's mechanosensors. These structures are composed of microfilaments and cytoplasm, covered by plasma membrane. Epithelial cell function is usually coupled to the density of microvilli and its individual size illustrated by diseases, in which microvilli degradation causes malabsorption and diarrhea. Atomic force microscopy (AFM) has been widely used to study the topography and morphology of living cells. Visualizing soft and flexible structures such as microvilli on the apical surface of a live cell has been very challenging because the native microvilli structures are displaced and deformed by the interaction with the probe. PeakForce Tapping® is an AFM imaging mode, which allows reducing tip–sample interactions in time (microseconds) and controlling force in the low pico‐Newton range. Data acquisition of this mode was optimized by using a newly developed PeakForce QNM‐Live Cell probe, having a short cantilever with a 17‐µm‐long tip that minimizes hydrodynamic effects between the cantilever and the sample surface. In this paper, we have demonstrated for the first time the visualization of the microvilli on living kidney cells with AFM using PeakForce Tapping. The structures observed display a force dependence representing either the whole microvilli or just the tips of the microvilli layer. Together, PeakForce Tapping allows force control in the low pico‐Newton range and enables the visualization of very soft and flexible structures on living cells under physiological conditions. © 2015 The Authors Journal of Molecular Recognition Published by John Wiley & Sons Ltd.     

Freeze-fracture study of taste bud pores in the foliate papillae of the rabbit

Summary In freeze-fractured specimens of taste buds from the foliate papillae of rabbits, the intercellular spaces are separated from the pore of the taste bud by zonulae occludentes of the tight-type. Below these tight junctions numerous desmosomes are found at irregular intervals. The epithelial cells adjacent to the pore are also joined by single strands of fusion. The microvilli arising from the neck of the type I cells have a high particle density. The microvilli of type II cells and especially the short microvilli of peripherally situated cells have a lower intramembranous particle density. The single microvillus of type III cells has a very large diameter and is longer than the other microvilli. It contains a few larger intramembranous particles and vesicle-like protrusions of the membrane facing the cytoplasm. Transverse fracturing reveals a filamentous fine structure in all microvilli. The physiological implications of these observations are discussed.Supported by grants from the Deutsche Forschungsgemeinschaft (Ja 205/5+6)     

Intrinsic mechanical properties of the extracellular matrix affect the behavior of pre-osteoblastic MC3T3-E1 cells

Mechanical cues present in the ECM have been hypothesized to provide instructive signals that dictate cell behavior. We probed this hypothesis in osteoblastic cells by culturing MC3T3-E1 cells on the surface of type I collagen-modified hydrogels with tunable mechanical properties and assessed their proliferation, migration, and differentiation. On gels functionalized with a low type I collagen density, MC3T3-E1 cells cultured on polystyrene proliferated twice as fast as those cultured on the softest substrate. Quantitative time-lapse video microscopic analysis revealed random motility speeds were significantly retarded on the softest substrate (0.25 ± 0.01 µm/min), in contrast to maximum speeds on polystyrene substrates (0.42 ± 0.04 µm/min). On gels functionalized with a high type I collagen density, migration speed exhibited a biphasic dependence on ECM compliance, with maximum speeds (0.34 ± 0.02 µm/min) observed on gels of intermediate stiffness, whereas minimum speeds (0.24 ± 0.03 µm/min) occurred on both the softest and most rigid (i.e., polystyrene) substrates. Immature focal contacts and a poorly organized actin cytoskeleton were observed in cells cultured on the softest substrates, whereas those on more rigid substrates assembled mature focal adhesions and robust actin stress fibers. In parallel, focal adhesion kinase (FAK) activity (assessed by detecting pY397-FAK) was influenced by compliance, with maximal activity occurring in cells cultured on polystyrene. Finally, mineral deposition by the MC3T3-E1 cells was also affected by ECM compliance, leading to the conclusion that altering ECM mechanical properties may influence a variety of MC3T3-E1 cell functions, and perhaps ultimately, their differentiated phenotype. bone; focal adhesion kinase; mechanotransduction; cytoskeleton; integrins     

Size-fractionated phytoplankton carboxylase activities in the Indian sector of the Southern Ocean

During the ANTARES 3 cruise in the Indian sector of the SouthernOcean in October–November 1995, the surface waters ofKerguelen Islands plume, and the surface and deeper waters (30–60m) along a transect on 62°E from 48°36'S to the iceedge (58°50'S), were sampled. The phytoplankton communitywas size-fractionated (2 µm) and cell numbers, chlorophyllbiomass and carbon assimilation, through Rubisco and ß-carboxylaseactivities, were characterized. The highest contribution of<2 µm cells to total biomass and total Rubisco activitywas reported in the waters of the Permanent Open Ocean Zone(POOZ) located between 52°S and 55°S along 62°E.In this zone, the picophytoplankton contributed from 26 to 50%of the total chlorophyll (a + b + c) with an average of 0.09± 0.02 µg Chl l^–1 for <2 µm cells.Picophytoplankton also contributed 36 to 64% of the total Rubiscoactivity, with an average of 0.80 ± 0.30 mg C mg Chla^–1 h^–1 for <2 µm cells. The picophytoplanktoncells had a higher ß-carboxylase activity than largercells >2 µm. The mixotrophic capacity of these smallcells is proposed. From sampling stations of the Kerguelen plume,a relationship was observed between the Rubisco activity perpicophytoplankton cell and apparent cell size, which variedwith the sampled water masses. Moreover, a depth-dependent photoperiodicityof Rubisco activity per cell for <2 µm phytoplanktonwas observed during the day/night cycle in the POOZ. In thenear ice zone, a physiological change in picophytoplankton cellsfavouring phosphoenolpyruvate carboxykinase (PEPCK) activitywas reported. A species succession, or an adaptation to unfavourableenvironmental conditions such as low temperature and/or availableirradiance levels, may have provoked this change. The high contributionof picophytoplankton to the total biomass, and its high CO₂fixation capacity via autotrophy and mixotrophy, emphasize thestrong regeneration of organic materials in the euphotic layerin the Southern Ocean.     

THE FINE STRUCTURE OF THE ABDOMINAL SENSE ORGANS OF PTERIOMORPHA (MOLLUSCA, BIVALVIA)

An ultrastructural study of the abdominal sense organs (ASO)of nine species, representing all three groups Of Pteriomorpha(Mytilina, Arcina, Pteriina), is offered. In all species investigated the sensory epithelium is high (40–130µm) and possesses two predominant cell types: (a) electron-densesupporting cells with microvilli only, pigment granules, andoval distal nuclei; (b) sensory cells with round proximal nucleiand electron-lucent plasma. Their narrow processes, always bunched,reach the surface bearing long (100–300 µm) stiffcilia. Each cilium has a polarized, orientated basal foot andseveral strong roots and is surrounded by nine specialized "stereo-microvilli"forming a basal plate in connexion with the basal body. Basalcontact of the sensory cells with nervous tissue proves theirreceptive nature. Structural uniformity as well as identical innervation provideevidence for the homology of the ASO in all Pteriomorpha. Thereis no homology between sensory cells and the similar, so-called"choanocyte-like cells".     

Neural Interaction between Cortical Taste Neurons in Rats: a Cross-correlation Analysis

In the rat cortical taste area (CTA), we recorded 31 pairs oftaste neurons and seven pairs of taste and non-taste neurons,with single or double electrodes. By using a cross-correlogram(CCG) in a stationary state, we examined the functional interactionbetween neurons of the pairs while activating them by tastestimulation. Though only 14.3% of the taste and non-taste neuronpairs were correlated, 54.8% of the taste neuron pairs showedcorrelated activities, 41.9% of them showing common inputs,including one with an additional excitatory connection. Theremainder (12.9%) showed excitatory connections with a timelag of 1–3 ms. When pairs were recorded using single ordouble electrodes with an intertip distance of <50µmin a dorsoventral direction, a larger fraction had correlatedactivities than when the intertip distance was >50 µm.Whereas pairs of neurons showed correlated activities in areaDI whatever the vertical intertip distance was, most of thepairs having correlated activities in area GI were found within50 µm of the vertical intertip distance. The taste profilesof common inputs to the pair were estimated on the basis ofpeak at time 0 in CCGs for various taste stimuli. The efficacycontribution of the source to target neurons tended to be largerwhen both had the same best stimulus. This tendency held truefor pairs showing excitatory connections. Interlayer excitatoryconnections were also evident. It is concluded that a functionalcolumn with a diameter of 50 µm may present in the CTAin rats, and that information flow is larger between pairs ofneurons with the same best stimulus. Chem. Senses 22: 517–528,1997.     

Annual variation in the abundance and size of heterotrophic nanoflagellates on the SW coast of Finland, the Baltic Sea

Annual variation and vertical distribution in the abundanceand cell volume of heterotrophic nanoflagellates (HNF) was studiedon the SW coast of Finland, the Baltic Sea. HNF cell numbersand mean cell volume varied annually from 90 to 10⁴ cells ml^–1,and from 3 to 32 µm³, respectively, with maxima in earlysummer. The proportion of choanoflagellates in the HNF communitywas 0–23%. Statistical analysis revealed the verticaldifferences in HNF abundance to be insignificant, but verticaldifferences in the size structure of HNF communities were found,especially during thermal stratification. The majority (>80%)of HNF were small (maximum dimension 2–4 µm); theproportion of large (>7 µm) cells were only 2–4%of the HNF abundance. An empirical equation for the relationshipbetween HNF cell length and volume is presented, and the measurementof flagellate volume by epifluorescence microscopy is discussed.     

Regeneration of Tissues in Wounded Stems: A Quantitative Study

When a dicotyledonous stem is wounded by longitudinally splittinga young internode into halves, cells near the cut surface proliferateto form a callus within which vascular tissues differentiateand tend to restore a vascular cylinder in each half. Threephases of regeneration after wounding were identified and quantifiedin stems of three Solanaceous species. (1) In an initial ‘lag’phase, lasting about 2 d, neither cell division nor enlargementwere detected, but mitotic figures were observed within about300 µm of the cut surface. (2) Throughout a second, ‘division’phase, from about days 2–10, cell division and enlargementoccurred. Both were initiated mainly in the two cell layersnearest the surface. A mass of callus formed, with new cellwalls mostly parallel to the surface. Cell enlargement laggedbehind cell division for the first few days, so that mean radialcell diameter decreased until day 6, thereafter remaining almostconstant at 30–40 µm. Towards the end of this phase,mitoses ceased within the callus except in the positions ofthe future vascular and cork cambia, where radial cell diameterfell towards a constant 15–20 µm. (3) During a third,‘differentiation’ phase, cell division was restrictedto the cambial zones, and derivatives differentiated into cork,phloem or xylem according to position. The rate of increasein cell number per transect was 1.5–2.0 cells d^–1,of which more than half was xylem. Capsicum annuum L., sweet pepper, Lycopersicon esculentum Mill., tomato, cambium, cell division, differentiation, regeneration, wounding of stems, xylem     

The Relationship Between Cell Size and Cell Number and the Distance from the Root Tip to the Region of Phloem Initiation in Groundnuts (Arachis hypogea. L. var. Bukene Red)

The effects of cell size and number on the distance from theroot tip to phloem inception in groundnuts have been investigated.The roots were treated with a number of plant growth regulatorshaving a wide range of effects on the growth rate. Increasing the growth rate of roots increased the distance ofphloem initiation and lignification from the apex. In all treatments,the average distance to phloem inception varied from about 590to 885 µm behind the root apex. The average lengths of the cortical and epidermal cells, whichwere 16 and 20 µm respectively, remained constant regardlessof the distance from the root tip.     

Marrow-derived stem cell motility in 3D synthetic scaffold is governed by geometry along with adhesivity and stiffness

Design of 3D scaffolds that can facilitate proper survival, proliferation, and differentiation of progenitor cells is a challenge for clinical applications involving large connective tissue defects. Cell migration within such scaffolds is a critical process governing tissue integration. Here, we examine effects of scaffold pore diameter, in concert with matrix stiffness and adhesivity, as independently tunable parameters that govern marrow‐derived stem cell motility. We adopted an “inverse opal” processing technique to create synthetic scaffolds by crosslinking poly(ethylene glycol) at different densities (controlling matrix elastic moduli or stiffness) and small doses of a heterobifunctional monomer (controlling matrix adhesivity) around templating beads of different radii. As pore diameter was varied from 7 to 17 µm (i.e., from significantly smaller than the spherical cell diameter to approximately cell diameter), it displayed a profound effect on migration of these stem cells—including the degree to which motility was sensitive to changes in matrix stiffness and adhesivity. Surprisingly, the highest probability for substantive cell movement through pores was observed for an intermediate pore diameter, rather than the largest pore diameter, which exceeded cell diameter. The relationships between migration speed, displacement, and total path length were found to depend strongly on pore diameter. We attribute this dependence to convolution of pore diameter and void chamber diameter, yielding different geometric environments experienced by the cells within. Bioeng. 2011; 108:1181–1193. © 2010 Wiley Periodicals, Inc.     

Identification of the Excitable Cells in the Petiole of Mimosa pudica by Intracellular Injection of Procion Yellow

Excitable cells in the petiole of Mimosa pudica were locatedby microelectrode technique and stained with Procion YellowMx4R which was previously filled in the electrode and injectediontophoretically into the cells. Microscopic observations ofsections of the stained petioles revealed that protoxylem parenchymacells and narrow phloem cells were excitable. The protoxylemlocalized just inside the metaxylem was composed almost entirelyof the parenchyma cells which were 106.3±5.2 µmlong (mean±EM, n=15) and 14.2±0.6 µm indiameter (n =33). The excitable phloem cells were 76.4±4.1µm long (n=7) and 7.0±0.3 pan in diameter (n=37)and were thought to be companion cells or narrow parenchymacells or both. Amplitudes of action potentials recorded fromthe petiolar surface had a linear relation to those from theexcitable cells in the same petiole. From this fact and thearrangement of excitable cells in the petiole, we conclude thatwhen the transmission of action potential takes place in thepetiole all excitable cells in it are activated. ¹ Present address: 1st Department of Physiology, Hamamatsu UniversitySchool of Medicine, Handa-cho 3600, Hamamatsu 431-31, Japan. (Received September 7, 1982; Accepted November 8, 1982)     

The Establishments of Tissue 4

Callus cultures of 12 temperate grasses were established, somefor the first time, by incubating detached roots or whole seedlingson a Linsmaier and Skoog basal medium which contained 2, 4-dichlorophenoxyaceticacid (2, 4-D) at 1.0 mg 1^–1 as the only growth hormone.The callus, which developed in the pericycle of the roots andon the embryo of the seeds, was subcultured on the same medium;further growth of the callus varied from good in the case ofDactylis glomeraia, Agrostis tennis, Cynosurus cristatus, andPoa trivialis, to poor in several Lolium species and varieties.Most cultures developed root primordia which sometimes grewinto visible roots, but shoot primordia, none of which grewinto shoots, were found only in the callus of Lolium multiflorumvar. westerwoldicum. Cell suspension cultures were also readily established and maintainedusing the same culture medium. Most cultures contained a highproportion of round or oval cells, which ranged from 18 to 77µm in diameter or length, while many also had a significantproportion of larger, more elongated cells which varied in lengthfrom 46 to 182 µm. The cells of Dactylis glomerata werecharacteristically larger and more convoluted than the cellsof other grass species that were examined. The addition of kinetinat 0.1 mg 1^–1 to the 2, 4-D-containing culture mediumincreased the proportion of irregular-shaped cells and reducedthe dispersion of the cells, perhaps by improving cellular contactand adhesion; in some species, such as Agrostis tenuis and Phleumpratense, the presence of kinetin promoted the deposition ofstarch granules in cells.     

Drought effects on cellular and spatial parameters of leaf growth in tall fescue

The effect of drought and recovery on cellular and spatial parametersof the growth process in tall fescue leaves was studied in twoexperiments. In both experiments plants grown on vermiculiteand maintained in a controlled environment were submitted toa 7 d drought period generated by withholding water. Droughtwas followed by a 3 d recovery period in experiment II. As leafelongation rate (LER) decreased during developing drought boththe growth zone length (initially 40 mm) and the maximum relativeelemental growth rate (initially 0.09 mm mm^–1 h^–1during the dark period of diurnal cycles) within the growthzone declined. But the growth zone still exhibited a lengthof approximately 15 mm when LER approached 0 under severe drought(–2.0 MPa predawn leaf water potential). The growth potentialof the basal 15-mm-long portion of the leaf was conserved duringthe period when drought effected the complete arrest of leafelongation. A (retrospective) analysis of the position-timerelationships of epidermal cells identified on leaf replicas(experiment II) indicated that the cell flux out of the growthzone responded very sensitively to drought. Before drought theflux was maximum at approximately 3.2 cells (cell file h)^–1during the dark period. Flux decreased to 0 when leaf elongationstopped. Flux also varied diurnally both under well-wateredand droughted conditions. In well-watered conditions it wasabout 30% less during the light than the dark period. Cell elongationwas also sensitive to drought. Under well-watered conditionsepidermal cell elongation stopped when cells attained a lengthof approximately 480 µm. During developing drought cellsstopped elongating at progressively shorter lengths. When LERhad decreased to almost nil, cells stopped elongating at a lengthof approximately 250 µn. When drought was relieved followinga 2 d complete arrest of leaf elongation then cells shorterthan 250 µm were able to resume expansion. Following rewateringcell flux out of the growth zone increased rapidly to and abovethe pre-drought level, but there was only a slow increase overtime in the length at which cell elongation stopped. About 2d elapsed until the leaf growth zone produced cells of similarlength as before drought (i.e. approximately 480 µm). Key words: Epidermal cell length, cell flux, (leaf) growth zone, leaf elongation rate, relative elemental growth rate, position-time relationships (path line, growth trajectory), drought, water deficit     

Isolation and characterization of a new,methylotrophic, acidogenic anaerobe,the marburg strain

The cellular characteristics of a new methylotrophic, acidogenic, anaerobic bacterium that was first isolated from a sewage digestor in Marburg, Federal Republic of Germany, is described. The Marburg strain is a mesophilic, Gram-positive, nonmotile, pleomorphic rod that performs homoacetic, homobutyric, or heteroacidic fermentations. Cell morphology varies from single or paired straight rods to rudimentary branched rods, club-shaped cells, or oval refractile cells. Cell heat resistance correlated with the presence of a few refractile cells. Electron micrographs of thin sections revealed a thick monolayered cell wall and an atypical spore structure. The DNA base composition was 48.8±0.2 mol% guanosine plus cytosine. Growth required factors in yeast extract; methanol, H₂/CO₂, glucose, fructose, lactate, and pyruvate were fermented as energy sources. Corrinoid levels varied from 0.35±0.16 to 7.9±1.6 μg/mg cell dry weight when cells 0.1% yeast extract, N₂/CO₂, 100 mM methanol, and 50 mM Na acetate displayed a 20h doubling time, finalA ₅₄₀ of 0.9, butyric acid yield of 25 mM, and ≈stoichiometry of 3 mol butyrate formed per 10 mol methanol fermented. The nameButyribacterium (emend.)methylotrophicum sp. nov. is proposed for the Marburg strain.     

Functional coupling in bovine ciliary epithelial cells is modulated by carbachol

The functional coupling of the ciliaryepithelium was studied in isolated pairs (couplets) of pigmentedciliary epithelial (PCE) and nonpigmented ciliary epithelial (NPCE)cells using the whole cell patch clamp and the fluorescent dye luciferyellow. One cell of the pair (usually the NPCE cell of a NPCE-PCE cell couplet) was accessed with a 2-5 M electrode, containing1-2 mM lucifer yellow, in the whole cell configuration of thepatch clamp. After voltage-clamp experiments were completed, cells were viewed under a fluorescent microscope to confirm that the cells werecoupled. The electrical coupling of the cells was also studied bycalculating the capacitance (using the time-domain technique), assuminga "supercell" model for coupled cells. The mean capacitance ofcoupled pairs was 79.8 ± 4.3 (SE) pF(n = 47) compared with single cellcapacitances of 36.8 ± 3.4 pF (n = 10) for PCE cells and 38.1 ± 3.1 pF(n = 15) for NPCE cells. Octanol,carbachol (CCh), and raised extracellularCa²⁺ concentration([Ca²⁺]_o)all caused uncoupling in pairs (couplets) of coupled NPCE and PCEcells. At room temperature (22-24°C), the capacitance of thecouplets decreased from 70.5 ± 8.0 to 48.0 ± 5.2 pF(n = 5) when exposed to octanol (1 mM), from 73.8 ± 9.2 to 43.2 ± 9.5 pF(n = 4) when exposed to CCh (100 µM), and from 80.5 ± 6.7 to 49.9 ± 7.8 pF(n = 4) when exposed to 10 mM[Ca²⁺]_o.The response to CCh was dose dependent; at higher temperatures of34-37°C, 10 µM CCh caused a 38% reduction in capacitance,from 53.7 ± 9.7 to 33.5 ± 3.3 pF(n = 7) with a half-time of 249 s, and100 µM CCh caused a 49% reduction in capacitance, from 51.3 ± 5.6 to 26.0 ± 2.4 pF (n = 7) witha half-time of 124 s. After pairs uncoupled and the uncoupling agentwas washed out, the cell pairs often exhibited an increase incapacitance that we interpreted as "recoupling" or a reopening ofthe gap junctional communication pathway; the half-time for thisprocess was 729 s after uncoupling with 100 µM CCh and 211 s afteruncoupling with 10 µM CCh. This interpretation was confirmedoptically by the spread of lucifer yellow into both cells of anuncoupled pair with a time course corresponding to the increase inelectrical coupling. The controllable coupling of ciliary epithelialcells extends the idea of a functional syncytium involved in activetransport. PCE cells take up solute and water from the blood, whichthen cross to NPCE cells via gap junctions and from there are secretedinto the posterior chamber of the eye. Modulation of the couplingbetween NPCE and PCE cells may provide a mechanism to controlsecretion.