Electron microscopic investigation of the surface membrane structures of the fat-cell and of their changes during depletion of the cell

Summary The main purpose of this paper was to investigate with the electron microscope the structural relationship between the fat cell and the surrounding connective tissue under various functional conditions and thereby to solve questions not decided in the past which concern the membrane of the fat-cell. Outside the well defined plasma membrane two layers were observed, one of lesser electron density next to the plasma membrane and another denser line which separates the former from the connective tissue ground substance. Fundamentally this three-layered surface membrane complex (Robertson) is the same as described by numerous authors in other cells bordering on connective tissue. However, the changes occurring in the surface membrane complex during depletion of fat cells are of special interest. The numerous long processes formed by the cell during the loss of fat in starvation are retracted in extreme depletion. At this time a pericellular space opens between the outer lamella and the plasma membrane. While the less dense material apparently becomes liquified the outer lamella of the surface membrane complex remains in contact with the connective tissue ground substance. These observations made it possible to interpret the surface membrane structures of the fat-cell as consisting, beside the plasma membrane, of a material derived from the ground substance, which is analogous to Robertson's gap substance at the surface of the Schwann cell, and of a limiting membrane toward the ground substance. The nature and possible derivation of the extracellular layers are discussed and the general functional significance of the surface membrane complex is emphasized. These considerations support the repeatedly raised objection against the use of the histological term basement membrane for the submicroscopic structures. During the depletion of the fat cell intensive micropinocytosis occurs regularly in the plasma membrane. It is suggested that the pinching off of numerous pockets may effect the elimination of membrane material in conjunction with the decrease in the surface area which has been found to take place in extreme depletion of the fat cell.This work was performed under the auspices of the U.S. Atomic Energy Commission.     

Changes in the distribution of integrins and their basement membrane ligands during development of human thyroid follicular epithelium

Interactions between cells and basement membrane components are crucial for the regulation of epithelial cell differentiation and polarization. We have studied by immunohistochemical methods the distribution of integrin adhesion proteins and some of their basement membrane ligands in foetal (16--19 weeks) and adult thyroid follicular epithelia. A diffuse immunoreactivity for only 3, v and 1 integrins was found in foetal follicular epithelium, whereas in adult follicular epithelium these integrins were expressed basally in a polarized manner. Additionally, 3 integrin was seen in a more basolaterally confined manner in adult follicular epithelium. Among basement membrane components, laminin 1, 1, 1 and 2 chains were found in epithelial basement membranes of the foetal thyroid gland, suggestive of the presence of laminins-1 and -3. In contrast, the basement membranes of adult follicular epithelium presented a much weaker immunoreactivity for the laminin 2 chain. Furthermore, immunoreactivity for the laminin 2 chain was occasionally seen in adult thyroid glands, apparently confined to myofibroblasts. Immunoreactivity for type IV collagen 1 and 2 (IV) chains was found in follicular basement membranes of foetal as well as adult thyroid gland. The results suggest that during maturation of foetal thyroid follicular epithelium a distinct polarization of integrins takes place. In mature thyroid follicular epithelium, the presumable adhesion-mediating integrin complexes are 31, v1 and/or v3 mediating adhesion to laminin-1 (1-1- 1) and type IV collagen trimer 12 (IV)     

X-ray microanalysis of calcium binding sites in Paramecium

Summary In Paramecium cells Ca⁺⁺-stimulated triggering of the exocytosis of secretory vesicles (trichocysts) was achieved by ionophores X-537 A or A 23187. Under triggering conditions electron dense deposits were present in some resting trichocysts and regularly in discharging trichocysts; upon subsequent fixation deposits occurred on the trichocyst membrane (on the inner side or within the membrane) and on the inner lamellar sheath from where deposits seemed to radiate into the secretory materials. Similar results were obtained with glutardialdehyde fixation alone which also triggers exocytosis but only at low concentrations. Element analysis by energy dispersive x-ray microanalysis ascertained the presence of Ca and P in deposits occurring in trichocysts. Those resting trichocysts which were devoid of deposits did not contain Ca or P enriched. Hence, an abrupt Ca⁺⁺-influx into individual trichocysts just before exocytosis seems to be involved in the triggering mechanism, possibly in combination with the sudden activation of an ATPase systemlocalized at those sites of the trichocysts which primarily contain the deposits. When paramecia were treated only with Ca⁺⁺ and then fixed with OsO₄ plus oxalate or merely with glutardialdehyde, electron scattering deposits were formed also on the inner side of the cell membrane and within the ciliary shaft (but rarely in trichocysts). Deposits obtained on cilia (including ciliary granule plaques) also contained Ca, P and S. Cells contain osmiophilic calcium-storing vacuoles which were selectively rich in Ca and S but devoid of P.     

Heterogeneity of epithelial cells in the human thymus

Summary To evaluate interrelationships among epithelial cells, and between morphology and function in the microenvironment, we studied the ultrastructural morphology of epithelial cells in sections of human thymus from donors aged 2 months to 31 years. Six types of epithelial cells were observed: subcapsular-perivascular (type 1); pale (type 2); intermediate (type 3); dark (type 4); undifferentiated (type 5); and large-medullary (type 6). Cells of types 2, 3 and 4 were found throughout the organ. The type-2 to -4 epithelial cells may represent various stages in a differentiation process. In this, type-2 cells are very active and type-4 cells are possibly degenerating elements. Type-4 cells can also contribute to Hassall's corpuscles. Type-5 cells were located mainly in the cortico-medullary region and showed the morphological characteristics of undifferentiated elements. Type-6 cells were located exclusively in the medulla and displayed characteristics of cellular activity. Small Hassall's corpuscles consisted of type-6 epithelial cells; in larger corpuscles many nuclei of type-6 cells were found. Cells of types 2 and 6 contained tubular structures (diameter approximately 20 nm).Concerning the function of thymus epithelial cells, the features associated with protein synthesis observed in cellular types 2 and 6 make them likely candidates for humoral factor-producing and/or secreting elements. In addition, type-2 and -3 cells in the cortex appear to contribute to a special pattern of epithelium-lymphocyte interaction (thymic nurse cells), as demonstrated by the intracytoplasmic location of lymphocytes in the epithelial cells. The various steps in intrathymic T-cell maturation occur at locations in a microenvironment composed of morphologically distinct epithelial cells.     

The β-1,3-glucan-binding protein from the crayfish Pacifastacus leniusculus,when reacted with a β-1,3-glucan,induces spreading and degranulation of crayfish granular cells

Summary A -1,3-glucan-binding protein (GBP) was purified from crayfish plasma, and incubated with laminarin (L), a -1,3-glucan. The GBP reacted with laminarin (GBP-L) induced strong spreading and partial degranulation of isolated and separated crayfish granular haemocytes. However, neither the GBP nor laminarin alone induced any changes in the crayfish granular cells. When monolayers of granular haemocytes were incubated with 20 g of GBP-L, more than 82% of the haemocytes were affected. The activity of GBP-L on granular cells was dose-dependent and a plateau was reached at 10 g of GBP-L. The degranulation of crayfish haemocytes induced by GBP-L seemed to occur by a regulated exocytosis, since it was strongly inhibited by specific blockers of this process such as SITS or calmidazolium. Monospecific anti-GBP antibodies also totally blocked the effect of GBP-L on crayfish granular cells. Indirect immunofluoresence staining demonstrated that the GBP-L could bind to the surface of granular cells, whereas GBP did not bind or bound very weakly to the haemocyte surface.     

Lipid profile of rat myelin subfractions

Myelin from adult rat brains was separated on a discontinuous sucrose gradient into three subfractions. Analysis of light, heavy and membrane fraction lipid classes was performed by HPTLC and densitometry while fatty acid composition was determinated by GLC. The more interesting results observed are: i) the membrane fraction resembles in its lipid and fatty acid composition other cell membranes (particulary oligodentrocytes); ii) light and heavy myelin are quite similar between them but the former has a higher content of sphingomyelin, a lower hydroxy/nohhydroxy cerebrosides ratio and a lower content of monoenoic fatty acids than the heavy subfraction. The results obtained could explain the different structures observed in each myelin subfraction since fatty acid composition, hydroxy fatty acids, sphingomyelin and cholesterol play a key role in the stability and structure of membranes.     

A potassium conductance activated by hyperpolarization in paramecium

Summary Voltage clamp studies show that the wild-type membrane ofParamecium tetraurelia contains a conductance component which is sensitive to hyperpolarization. This component manifests itself as anomalous, or inward going, rectification of membrane voltage in response to applied constant current pulses and as a hyperpolarizing spike when no K is added to the external solution (Y. Satow, C. Kung, 1977.J. Comp. Physiol. 11999). Like the conductances which underlie anomalous rectification in other cells, the hyperpolarization-sensitive conductance inParamecium is specific for K, and the magnitude of the voltage-dependent conductance change depends not only on voltage but also on external potassium concentration. The internal potassium ion concentration ofParamecium is calculated to be between 17 and 18mm.     

On the sufficiency of the velocity field for perception of heading

All models of self-motion from optical flow assume the instantaneous velocity field as input. We tested this assumption for human observers using random-dot displays that simulated translational and circular paths of movement by manipulating the lifetime and displacement of individual dots. For translational movement, observers were equally accurate in judging direction of heading from a velocity field with a two-frame dot life and a direction field in which the magnitudes of displacement were randomized while the radial pattern of directions was preserved, but at chance with a speed field in which the directions were randomized, preserving only magnitude. Accuracy declined with increasing noise in vector directions, but remained below 2.6° with a 90° noise envelope. Thus, the visual system uses the radial morphology of vector directions to determine translational heading and can tolerate large amounts of noise in this pattern. For circular movement, observers were equally accurate with a 2-frame velocity field, 3-frame acceleration displays, and 2-frame and 3-frame direction fields, consistent with the use of the pattern of vector directions to locate the center of rotation. The results indicate that successive independent velocity fields are sufficient for perception of translational and circular heading.     

An argyrophil fibrillar system and amitotic nuclear division in pars intermedia cells of the rainbow trout (Salmo irideus)

Summary Argyrophil fibrils were revealed in pars intermedia chromophobe cells of Salmo irideus. For the light microscopical demonstration of the fibrils a recently developed copper sulphate-silver protein technique for Bouin-fixed hypophyses was used.The fibrils, apparently belonging to one fibrillar system, are found only in limited regions of the cytoplasm. They occur in ring-, loop-, or cracknel shapes in the close vicinity of the rounded nucleus in the chromophobe cell, and continue in linear shape into the cell base or cell apex where they may end near the adjacent basement membrane.Electron microscopically, the fibrils are composed of filaments, 70–90 Å in diameter, which are arranged in parallel. Bundles of filaments are frequently found near the nucleus. In addition to perinuclear filaments, bundles of filaments with tiny offshoots occur in different areas of the cytoplasm, extending sometimes as far as the cell membrane which is located near the basement membrane of the pars intermedia epithelium.In cells with a bi- or trilobular, apparently amitotically dividing, nucleus a fibrillar loop or ring usually surrounds the interlobular part(s) of the nucleus. This relation of the fibrils to the dividing nucleus and their consisting of regularly arranged filaments indicates the significance of the fibrillar system in the chromophobe cell. It is thus suggested that the fibrillar system is involved in the constriction of the nucleus during amitosis.The number of chromophobe cells in which fibrils are light microscopically visible varied greatly among the rainbow trout used.Miss M. C. Wentzel is thanked for her technical assistance, Mr. M. Veenhuis for his advice and excellent help with the preparation of the electron micrographs, and Mr. L. Hoekstra for preparing the final drawings. Mr. N. J. Williams' corrections to the English are gratefully acknowledged.     

The effect of ferrous sulphate on the yield and manganese uptake of oats on sandy soil fertilized with pyrolusite

Summary In pot experiments with oats on a sandy soil deficient in managanese and with an extremely low content of reducible managanese, the effect of 0.125 g reducible managanese in two pyrolusite fertilizers (-managanese dioxides) made in 1938 and 1955, respectively, was compared with the effect of 0.125 g water-soluble managanese as managanese sulphate.On soil to which no ferrous sulphate was added, the response to manganese added as Pyrolusite 1938 and Pyrolusite 1955 was of the same magnitude, but slightly less than the response to managanese added as managanese sulphate. The difference between the effect of pyrolusite and that of managanese sulphate was not, however, statistically significant.On soil to which ferrous sulphate had been added, a response was obtained to Pyrolusite 1938 twice as high, and to Pyrolusite 1955 three times as high as the response to manganese sulphate. These results support the suggestion² that the manganese effect of ferrous sulphate is due to its ability to reduce higher managanese oxides in the soil.Since soil dressed with ferrous sulphate gave a response to added Pyrolusite 1955 which was about 50 per cent higher than the response to Pyrolusite 1938, and since, furthermore, the same amount of reducible manganese and the same number of manganese atoms on the surface of the two fertilizers were involved, it is probable that the difference between the effects of the two pyrolusite fertilizers found under these circumstances is due to difference in their crystallinity.     

Fusion of cultured dog kidney (MDCK) cells: I. Technique,fate of plasma membranes and of cell nuclei

Summary The evaluation of the intracellular signal train and its regulatory function in controlling transepithelial transport with electrophysiological methods often requires intracellular measurements with microelectrodes. However, multiple impalements in epithelial cells are hampered by the small size of the cells. In an attempt to avoid these problems we fused cells of an established cell line, Madin Darby canine kidney cells, originally derived from dog kidney, to giant cells by applying a modified polyethylene glycol method. During trypsin-induced detachment from the ground of the petri dish, individual cells grown in a monolayer incorporate volume and mainly lose basolateral plasma membrane by extrusion. By isovolumetric cell-to-cell fusion, spherical giant cells are formed within 2 hr. During this process a major part of the individual cell plasma membranes is internalized. Over three weeks following cell plasma membrane fusion degradation of single cell nuclei and cell nuclear fusion occurs. We conclude that this experimental approach opens the possibility to investigate ion transport of epithelia in culture by somatic cell genetic techniques.     

Glycoconjugates of the Rat Ciliary Body Epithelium: A Lectin Histochemical and Protein Blotting Study

The present study sought to identify and partially characterize the glycoconjugates specific to the double-layered ciliary body epithelium of the rat eye by lectin histochemistry and lectin blottings. Hydrated paraffin sections of Carnoy-fixed Sprague-Dawley rat eyes were stained with a panel of 21 different biotinylated lectins, followed by streptavidin-peroxidase and the glucose oxidase-diaminobenzidine-nickel staining procedure. The results of lectin histochemistry revealed that the inner epithelial layer was rich in GlcNAc(1,4)GlcNAc, -Gal, Gal(1,3)GalNAc, GalNAc(1,3)GalNAc/Gal, GalNAc(1,6)Gal, Fuc(1,2)Gal(1,4)GlcNAc and Gal(1,4)GlcNAc(1,2)Man(1,6) sugar residues as shown by its positive reactivities with S-WGA, PWA, DSA, GS-I-B4, PNA, DBA, SBA, WFA, UEA-I, LTA and PHA-E. The reactivities of GS-I-B4, PNA, DBA and SBA were restricted to the inner layer at the tips of the ciliary processes. On the other hand, the outer epithelial layer was stained evenly by DSA and Jacalin, and partly by MAA, showing that this epithelial layer was rich in GlcNAc(1,4)GlcNAc, Gal(1,3)GalNAc and NeuAc(2,3)Gal disaccharides. These lectin binding patterns of the ciliary body epithelium suggest a topographical and functional difference in this double cell-layered epithelium. Their possible roles in the secretion of aqueous humour and production of ciliary zonule are discussed. Some identified lectin markers specific to these two cell layers may be useful for further experimental studies. Glycoproteins extracted from the dissected ciliary body were separated by SDS-PAGE electrophoresis and analyzed by protein blottings with 8 different lectins. The results showed that at least 10 major membrane-bound glycoproteins, with molecular weights ranging from 30 to 150kD, rich in -GlcNAc, -Gal, /-GalNAc and NeuAc(2,6)Gal residues, were present in the microsomal fraction.     

Ultrastructure of the surface of the epithelial cells in the rat retina

Summary The epithelial cells in the retina of rats were examined by scanning and transmission electron microscopy.The apical surface of the epithelial cells is covered by a large number of microvilli, which are embedded in an extracellular material. Distinct holes remaining after detached photoreceptor cell segments are numerous. The cells are polyhedronal, usually hexagonal and holes are scattered in their plasma membrane. Many of the epithelial cells are binucleated. The borders between adjacent cells are always close to each other. Numerous cytoplasmic folds or foot processes occur at the base of the cells as well as an extracellular space.The characteristic surface structures are discussed in relation to the function of the retinal epithelial cells.Supported by grants from the Swedish Medical Research Council (B70-12X-2543-02) and Riksföreningen mot Cancer (69171, 265-B69-01X).     

Anomeric preference of glucose utilization in rat erythrocytes

The -anomer of glucose relative to the -anomer was more rapidly metabolized into lactate by rat erythrocytes at 37° C (/ ratio = ca. 1.3): the amounts of - and -D-glucose metabolized into lactate during 3 min were 0.21 and 0.27 mol/gHb, respectively. Also, the transport of -D-glucose into erythrocytes was more rapid than that of -D-glucose: the amounts of - and -D-glucose transported into erythrocytes during 3 min were approximately 3.5 and 5.0 mol/gHb, respectively. Glucose phosphorylation by rat erythrocyte hexokinase (i.e., a possible rate-limiting step in glycolysis) occurred at higher velocities with the -anomer than with the a-anomer (/ ratio = 1.28). The Km value of hexokinase for either anomer of glucose was 53 M. The glucose concentrations in erythrocytes incubated with - and -D-glucose reached about 1 mM in 1 min, indicating that hexokinase is almost completely saturated with glucose within less than 1 min. The results suggest that glucose phosphorylation and glucose transport are major and minor determinants, respectively, for the anomeric preference of glucose utilization in rat erythrocytes.     

Development of high frequency multiple shoot formation in Persian clover (<Emphasis Type="Italic">Trifolium resupinatum</Emphasis> L.)

An efficient and reliable micropropagation system for Persian clover (Trifolium resupinatum L.) was developed using different explants and media. Node, hypocotyl and cotyledonary node explants were cultured on Murashige and Skoog (MS) medium supplemented with combinations of either 6-benzyladenine (BA) and indole-3-butyric acid (IBA) or BA, Kinetin (KIN) and IBA. Direct multiple shoots developed within 6weeks in all explants in most media tested. The best shoot multiplication capacity was obtained from cotyledonary node explants on MS medium containing 7.1M BA and 1M IBA or 14.1M BA and 1M IBA. Elongated shoots were rooted on either MS medium alone or combination with different concentrations of indole-3-butyric acid (IBA), indole-3-acetic acid (IAA) and -naphthaleneacetic acid (NAA). High rooting was achieved in half strength MS medium containing 8M IBA.     

The air-blood barrier in the human lung

Summary The air-blood barrier was studied in replicas of freeze-fractured lung biopsies collected from healthy human subjects. Adjacent pneumocytes display a belt-like network composed of 3–7 superimposed ridges (fibrils) on the P face and complementary grooves on the E face, i.e., a structure corresponding to a tight junction. On the other hand, adjacent capillary endothelial cells show a continuous system of 2–4 membrane foldings. These appear mainly as smooth surfaced crests on the P face; on the E face furrows are seen, at the bottom of which a row of particles is situated. This arrangement suggests a leaky type of junction. Discontinuous occluding junctions are located in the pericytic venular segment of the alveolar vessels. The present findings are in agreement with previous physiological and ultrastructural tracer studies locating the main part of the diffusion barrier for small polar solutes and proteins in the alveolar epithelium. Communicating junctions are demonstrated between type I and type II pneumocytes, indicating intercellular cooperation between these cells of common embryonic origin, but which fulfill different functions in the adult. In the endothelium of the non-muscular alveolar vessels communicating junctions are lacking. Desmosomes occur in the epithelium between type I and type II pneumocytes; square arrays of particles characterize the plasma membrane of type I pneumocytes.A portion of this work was presented in partial fulfillment for the degree of Dr. med., Hannover Medical School     

Plasma membrane display of anti-viral single chain Fv fragments confers resistance to tobacco mosaic virus

We tested the hypothesis that membrane-anchored anti-viral antibodies can confer viral resistance to transgenic plants. A heterologous expression system was developed for plasma membrane targeting of anti-viral antibodies using mammalian transmembrane domains. A tobacco mosaic virus (TMV) neutralizing single-chain Fv antibody fragment (scFv24) was targeted to the endoplasmic reticulum and integrated into the plasma membrane of tobacco cells, using mammalian signal peptides and membrane receptor transmembrane domains. The human platelet-derived growth factor receptor (PDGFR) transmembrane domain or the T-cell receptor -domain (TcR) transmembrane domain was fused to the C-terminus of TMV-specific scFv24 to target expression of scFv24 as an extracellularly facing plasma membrane protein. Western blot and ELISA analyses were carried out to confirm functional expression of the recombinant fusion proteins scFv24-PDGFR and scFv24-TcR in transgenic tobacco suspension cultures and transgenic plants. Immunofluorescence and electron microscopy showed that the TcR transmembrane domain targeted scFv24 to the tobacco plasma membrane. Bioassays of viral infection showed that transgenic tobacco plants expressing scFv24-TcR were resistant to TMV infection. These results demonstrated that membrane anchored anti-viral antibody fragments are functional, can be targeted to the plasma membrane in planta and are a novel approach for engineering disease-resistant crops.     

Triggering and predisposing factors in the “Red” decline syndrome of Norway spruce (Picea abies)

Summary Analysis of 62 mature Norway spruce (Picea abies provenance Viborg) trees growing in a Danish plantation was undertaken along with analysis of their nutrient contents (N, P, K, Ca, Mg, Fe, Mn, Cu, Zn, B and Na), in each of the three youngest needle age classes, from branches of four exposure directions near the tree top. The aim was to investigate if one among the studied possible predisposing factors was also a triggering factor in the 1989 outbreak of the Red Norway spruce decline in Denmark. Neither nutrient imbalance or deficiency, nor excessive N-deposition or salt-stress were indicated as triggering factors in 1989. The Red syndrome, noticeable for the bright red colour of the current-year needles, was found to be an extension of the European type Novel Decline. Red syndrome is similar to previously reported phenomena of top-dying and sub top-dying, in that it had fewer needle age classes and significantly higher contents of mobile cations (and Ca) in the younger needle classes. Tree ring analysis suggested that the Red syndrome was initiated in the early 1980s, when the trees experienced adverse climatic conditions. Because of this long-term development of the Red Norway spruce decline syndrome, it is concluded that a triggering factor is of minor importance relative to the multitude of predisposing factors.     

Interaction of alpha-lactalbumin with mini-alphaA-crystallin

A-Crystallin can function like a molecular chaperone. We have recently shown that residues 71-88 in A-crystallin represent the chaperone active site of the protein. A peptide containing the sequence of A-crystallin sequence DFVIFLDVKHFSPEDLTVK (mini A-crystallin) by itself displays the antiaggregation property of A-crystallin. We have prepared a complex of reduced -lactalbumin and mini-A-crystallin and investigated the nature, conformation, and properties of the complex by dynamic light scattering, HPLC analysis, CD spectroscopy, and fluorescence studies. Although mini-A was able to prevent the precipitation of reduced -lactalbumin, large aggregates (50-500 nm) of the complex were formed during the assay. Amino acid composition estimation revealed that -lactalbumin and mini-A-crystallin were present in 1:2 ratio in the aggregates. During our study significant red shift in the Trp fluorescence emission maximum and an increase in Bis-ANS binding to the mini A-crystallin-bound -lacatalbumin were observed. The CD spectra of the complex showed a significant loss of -helical content but the -sheet content appeared to be less affected, indicating the molten-globule state of the reduced lactalbumin in the complex. These data show that the active site of A-crystallin by itself can maintain a significantly denatured and unfolded protein in soluble form.     

Field work in Romania: Introduction

Conclusions An even broader theoretical conclusion is in order. The term modernization has been enshrined in the value-free literature on development which the Western academy has created and supported. But the experience of Brasov, a local manifestation of Romanian development, makes it clear that modernization is by no means confined to Western assumptions, and that it always occurs in specific ways depending on the social situation of the society undergoing change, and on the political nature of the program being adopted. In short, socialist modernization in Romania is not parallel to modernization under capitalist auspices in analogous areas.