bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitative aspects of proliferation in the nasal epithelium of the rhesus monkey embryo
Authors: Wilson Doris B. , Hendrickx Andrew G.
Published: April 2021

Proliferation was studied in the nasal epithelium of rhesus monkey embryos ranging in age from 26 through 41 days of gestation (stages 13 – 20). Labeling indices were tabulated 1 h after an intrachorionic injection with [3H]thymidine, and the distribution of labeled cells was determined at intervals ranging from 1 h to 11 h after injection. The labeling index showed a chronological decrease from 69·4% in the nasal placode at stage 13 to 31·6% in the nasal pit at stage 20. The vomeronasal epithelium was distinctive in that the basal cells lost their ability to incorporate [3H]thymidine as early as stage 18. In the remainder of the nasal epithelium, mitotic figures were initially confined to apical regions but later occurred in basal regions as the embryonic pattern of proliferation became converted to the adult type.
An ultrastructural and biochemical study of high density primary cultures of embryonic chick chondrocytes
Authors: Oakes B. W. , Handley C. J. , Lisner F. , Lowther D. A.
Published: April 2021

Primary cultures of embryonic chick chondrocytes inoculated at high densities (5 – 6 × 106 cells/25 cm2 Falcon Flask) with adequate pH control, grow rapidly and multilayer with 5 – 8 cell layers forming a uniform thick approximately 50 – 80 μ m dense cartilage matrix. Cultures inoculated at lower densities tended to form nodular aggregates of chondrocytes secreting a typical cartilage matrix with the internodular areas occupied by elongated, vacuolated, fibroblast-like cells secreting little matrix. Ultrastructurally, using ruthenium red during fixation, early cultures revealed rounded cells surrounded by a collagen matrix of small diameter (150 nm) collagen fibres to which were irregularly attached electron-dense granules approximately 20 nm diameter. Fine filaments of 1 nm diameter linked the electrondense granules to one another, to collagen fibres and to the cell membrane. In older cultures larger cells were observed with ultrastructural features similar to hypertrophic chondrocytes seen in vivo. Electron-dense material composed of fine needle-like structures was observed in the matrix associated with these cells. These observations were interpreted as early calcification occurring in vitro in relation to hypertrophic cells.

The molecular weight of the proteoglycans and the spectrum of glycosaminoglycans synthesized were similar to that of normal embryonic chick cartilage, demonstrating maintenance of chondrocyte differentiation in this system.

This study demonstrates that it is possible by using high density cultures with adequate nutrient media and pH control to maintain chondrocytes in a differentiated state using the criteria of ultrastructural morphology and specificity of the matrix they synthesize.
Mouse teratomas and embryoid bodies: their induction and differentiation
Authors: Iles S. A.
Published: April 2021

Teratomas were induced by the transfer of mouse blastocysts (C3H and 129/J strains) and egg-cylinders (C3H) to extra-uterine sites. C3H and 129/J blastocysts cultured in vitro for 4 or 5 days could also form teratomas in extra-uterine sites.

Four transplantable teratomas, or teratocarcinomas, were derived from C3H embryos; embryoid bodies were derived from each line. The differentiative capacity of a teratocarcinoma was shown to be similar whether it was maintained as a solid tumour or as embryoid bodies.
Retino-tectal projections from half-ventral and half-dorsal eye rudiments in Xenopus
Authors: Feldman Joan D.
Published: April 2021

When the ventral half of a developing eye in Xenopus larvae was removed at stage 32, the remaining fragment rounded up and developed into an eye which looked macroscopically normal by mid-larval stages. Eyes from half-dorsal rudiments were usually small, had more than one ventral fissure, and showed abnormal pupils. The contralateral retinotectal projection was always found to be normally ordered when mapped in later tadpole stages, or, just after metamorphosis. No mirror-image duplicated maps were seen, as was found pre-viously in eyes deriving from half-nasal and half-temporal rudiments.

It is concluded that the ‘rule of distal transformation’ does not apply to eyes which are generated from embryonic rudiments.
Identification and distribution of gap junctions in the mesoderm of the developing chick limb bud
Authors: Kelley Robert O. , Fallon John F.
Published: April 2021

Sub-ridge, core, anterior and posterior borders of mesoderm were dissected from stages 22-24 chick wing buds to investigate whether structures for intercellular coupling develop between mesenchymal cells. Fine structure was examined using techniques of transmission electron microscopy, freeze-fracture and scanning electron microscopy. Gap (communicating) junctions which were observed between mesenchymal cells of all limb bud regions were distributed between apposed cell bodies, points of contact between cell processes and other cell bodies, and between contacting tips of slender cell projections. In addition, particularly in the sub-ridge region, filopodia were observed to extend through the intercellular matrix to contact other cells several micrometers distant.

The observations reported in this paper show that mesodermal cells throughout the limb have the structural capability for electrotonic and metabolic coupling during a critical period of morphogensisis in the avian limb. Whether intercellular signals which are thought to be transmitted through gap junctions are active in normal limb development remains to be investigated.
The development of projections and connections from transplanted locust sensory neurons
Authors: Anderson Hilary
Published: April 2021

Neurons innervating wind-sensitive hairs on the locust head form characteristic projections and connections within the CNS. These depend on intrinsic properties of the epidermis from which the hair and its neuron are formed (Anderson & Bacon, 1979; Bacon & Anderson, 1984). To investigate further these intrinsic properties and also extrinsic factors involved in guiding axon growth and determining synaptic connectivity, pieces of epidermis from the head were transplanted to the posterior head, prothorax, or mesothorax. Thus wind-sensitive neurons developing from the grafts were caused to grow into foreign parts of the CNS.

The neuronal projections from the graft hairs were examined by filling the axons with cobalt, and their connectivity with an identified interneuron, the Tritocerebral Commissure Giant, was examined by recording electrophysiologically the activity of the interneuron during stimulation of the graft hairs.

The results show that 1) the neuronal projections are confined to one tract, the median ventral tract, and to one arborization area, the ventral association centre, in all ganglia; 2) in all ganglia, neurons from different epidermal regions preserve their location-specific properties of forming ipsilateral or additional contralateral projections; 3) the extent of their projection in the CNS is not interpretable in terms of intrinsic instructions only; 4) in foreign ganglia, they fail to form connections with their normal target interneuron.
On the Normal and Abnormal Development of the Feather
Authors: Cohen J. , ’Espinasse Paul G.
Published: April 2021

A brief résumé is given of previous views of feather development. It is here emphasized that the collar is not to be regarded as producing a feather, but that it is in its entirety transformed into a feather and so is lost each time a feather is plucked. The culture of collar fragments is described. Many cultures produced apparently normal barbs and barbules without any process resembling concrescence. It is concluded that the only movement of tissue which occurs during the transformation of a collar into a feather is along the generators of the feather cylinder. The growth process concerned in the transformation of collar into feather is thus an essentially simple one of elongation such that distal collar becomes distal feather and proximal collar becomes proximal feather. It is the original length from distal collar to proximal collar that increases to become the whole length of the feather. Thus the structure of a particular feather may be projected back in a simple manner and the structure of the collar which it once was can be deduced. The collar, however, is indeed produced from the papillar ectoderm and the manner of its production is considered as a topological problem. A feather with a distal abnormality but which is normal proximally, resulting from a collar that is abnormal distally but normal proximally, may be repeated. The papilla in this case must return to a condition of abnormality after producing the normal proximal part of each such collar in order subsequently to produce another collar with a similar abnormality distally. From the results of papilla transplant experiments described here and from those reported from other workers, it is deduced that the manner of the production of a collar from a papilla involves a preferential growth of dorsal ectodermal tissue : tissue which was dorsal on the papilla grows over the top and down the ventral side of the dome produced at the top of the healing papilla. Some evidence is presented that this occurs both when a pin-feather is plucked and when a feather matures normally by closure of its inferior umbilicus. ‘Longitudinal’ and ‘transverse’ chimaerae, double feathers and feathers with defective apices, and their repetition, are considered and explained in terms of our views.
An Analysis of the Postgastrula Differentiation of the Hypomere
Authors: Finnegan Cyril V.
Published: April 2021

When a small mass of hypomeric mesoderm is cultured in vitro within an ectodermal ball (M series) the splanchnic portion is collected in the centre and some haematopoiesis (erythropoiesis) occurs. In explants from post-stage 18 explants, cultured beyond two weeks, granulopoiesis was found in a differentiating reticulo-endothelium. When a group of endoderm cells were added to the explanted hypomere (M+E series) the younger postgastrula stages (stages 13–18) now demonstrated a histogenesis similar to the older stages in the M series (above), while the poststage 18 explants undergo a similar histogenesis but more rapidly than in the previous series. Some of these older-stage explants gave evidence Of axial orientation in the splanchnic mesoderm. When the mass of hypomere was increased (2M series) a large amount of peritoneum and endothelium was produced in all explants along with the usual erythropoietic area. No reticulo-endothelium or granulopoiesis appeared. The explants from the older donors (stages 17–20) frequently indicated an axial pattern in the splanchnic mesoderm. When groups of endoderm cells were added to the increased mass of hypomere (2M+E series) the early postgastrula explants (stages 13–16) evidenced an axial pattern for the first time in the endothelial-haematopoietic histogenesis of the splanchnic mesoderm. Again, no reticulo-endothelium with granulopoiesis was observed. Analysis of mitotic counts in the various explants indicated that the endoderm was influencing differentiation rather than growth (i.e. cell-division). Similarly, the increase in mass of hypomere appeared to influence differentiation and also to stabilize growth. The experimental results are considered to further indicate the synergistic role of the endoderm in the histogenesis of the splanchnic hypomere.
Influence inductrice de l’encéphale et de la chorde sur la morphogenèse du squelette cranien chez l’embryon de Poulet
Authors: Schowing par Jean
Published: April 2021

De nombreux auteurs, parmi lesquels Parker (1876) et de Beer (1937), ont étudié et décrit le développement de la tête chez l’embryon d’Oiseau. C’est en 1933 que P. Ancel et Ét. Wolff ont inauguré l’analyse expérimentale des facteurs qui président à l’organogenèse embryonnaire chez le Poulet. Les travaux d’Ét. Wolff (1936) sur les bases de la tératogénèse expérimentale chez les Vertébrés amniotes ont incité d’autres collaborateurs à des recherches sur la morphogenèse des différents organes de l’embryon.
Experimental Investigations of Morphogenesis in the Growing Antler
Authors: Goss R. J.
Published: April 2021

Investigations of the morphogenesis of antler growth have been undertaken in the sika deer (Cervus nippon). Operations were performed on the left antlers, while the intact right antlers served as controls. Amputation of the terminal centimetre of growing antlers seriously impedes further development except for the production of an abortive outgrowth from the stump. By contrast, transverse amputation through the middle of the pedicle is followed by normal antler formation. Removal of the entire pedicle permits antler development to occur, although at a retarded rate. Therefore, antlerforming potentiality resides in the growing tips of the elongating antler as well as in the tissues of the entire pedicle. The extent of morphogenetic regulation for defects surgically inflicted on the antler bud or pedicle depends on the age of the antler and the location of the injury. Excision of the posterior halves of 1-inch antler buds results in corresponding deficiencies in the antlers produced. The same injury to an extremely rudimentary bud, however, is followed by considerable regulation, and removal of the posterior half of the pedicle prior to shedding of the old antler has no effect on the normal development of the resulting antler. Ablation of anterior or median halves of pedicles likewise causes no morphogenetic abnormality, but if the lateral half of the pedicle is removed no antler develops. The importance of the lateral half of the antler-forming structure is further illustrated by the fact that a complete and normal antler can grow from just the lateral half of a pedicle or of a 1-inch antler bud. The morphogenetic field of the antler can give rise to double antlers if bisected longitudinally; transverse subdivision, however, does not alter subsequent normal production of a single antler. It is proposed that a relationship between the initial mass of antler-forming material and the degree of morphogenetic expression may possibly exist.
miRNA interplay: mechanisms and consequences in cancer
Authors: Hill Meredith , Tran Nham
Published: April 2021

Canonically, microRNAs (miRNAs) control mRNA expression. However, studies have shown that miRNAs are also capable of targeting non-coding RNAs, including long non-coding RNAs and miRNAs. The latter, termed a miRNA:miRNA interaction, is a form of self-regulation. In this Review, we discuss the three main modes of miRNA:miRNA regulation: direct, indirect and global interactions, and their implications in cancer biology. We also discuss the cell-type-specific nature of miRNA:miRNA interactions, current experimental approaches and bioinformatic techniques, and how these strategies are not sufficient for the identification of novel miRNA:miRNA interactions. The self-regulation of miRNAs and their impact on gene regulation has yet to be fully understood. Investigating this hidden world of miRNA self-regulation will assist in discovering novel regulatory mechanisms associated with disease pathways.
DMM Outstanding Paper Prize 2020 winner: Sarah Colijn
Authors: Hackett Rachel
Published: April 2021

Disease Models & Mechanisms (DMM) is delighted to announce that the winner of the DMM Prize 2020 is Sarah Colijn, for her paper entitled ‘Cell-specific and athero-protective roles for RIPK3 in a murine model of atherosclerosis’ (Colijn et al., 2020b). The prize of $1000 is awarded to the first author of the paper that is judged by the journal's editors to be the most outstanding contribution to the journal that year. To be considered for the prize, the first author must be a student or a postdoc of no more than 5 years standing.
The effects of starvation and light on intra-mitochondrial granules in Physarum Polycephalum
Authors: Nicholls T. J.
Published: April 2021

The presence of spherical or rod-shaped, electron-dense particles was noted within the mitochondrial cristae of Physarum polycephalum plasmodia. When sporulation was stimulated by starvation and subsequent illumination it was found that the numbers of such particles present per mitochondrion increased during the starvation phase, reaching a maximum after about 4 days, and then decreased rapidly during exposure of the plasmodium to light. Few particles were present in the mitochondria of spores. It is suggested that the particles may be similar to the mitochondrial granules of other organisms in providing a repository of calcium, which in Physarum is deposited in the capillitium during sporulation.
Surface extensions on bhk cells grown in monolayers and agar suspension
Authors: Tuffery A. A.
Published: April 2021

Three types of surface extensions found on a subline of BHK cells capable of growing in agar are described.

These are:Microvilli-borne on the cell surface, lacking rigid cores, found on trypsinized cells and on cells growing in solid tissue type growth;Microspikes-borne on the cell margins, containing rigid cores, found on spread, attached cells, but not trypsinized cells or cells growing in solid tissues;Retraction fibrils-coreless ‘passive’ cell extensions drawn out distally as motile cells move across a surface.

Microvilli also seem to be associated with micropinocytic vesicles. It is suggested that microvilli on tissue cells in vitro may be homologous with the cell membrane interdigitations found in, for example, gut epithelium, and possibly also with the microvilli of brush borders. Their association with these structures suggests they may play a role in producing a local environment near the cell membrane in which micropinocytosis may function more effectively.
An inhibitor present in calf serum which prevents growth of BHK21 cells in suspension culture
Authors: Otsuka H.
Published: April 2021

BHK21 cells do not form colonies in soft agar suspension culture in the presence of calf serum; but if swine serum is used instead they do so. In methylcellulose suspension culture supplemented with calf serum the cells synthesize very little RNA or DNA, but if swine serum is used there is a marked increase of syntheses. Calf serum was found to contain an inhibitor which prevents the induction, but not the continuation, of RNA and DNA synthesis in BHK21 cells. In contrast the growth of polyoma-virus-transformed cells is not affected by this serum inhibitor which has been partially purified from calf serum by ammonium sulphate precipitation followed by Sephadex G-200 column chromatography.
Brain extracellular space and membrane morphology variations with preparative procedures
Authors: Schultz R. L. , Karlsson U. L.
Published: April 2021

When considering extracellular space and plasma membrane structure in the brain, duration of postfixation in osmium tetroxide and embedding procedure are equally as important as the dehydrating agent employed. The use of ethanol instead of acetone for dehydration does not adequately explain the finding of a 5–30 nm space instead of apposed membranes. In this investigation it was found that postfixation for 3 h stabilized the tissue sufficiently so that ethanol or acetone dehydration followed by Vestopal W, Epon 812, or Araldite 502 embedding produced similar results, and apposed, triple-layered plasma membranes were prevalent. In contrast, when postfixed for a shorter time, namely 90, 45 or 30 min, ethanol dehydration followed by Epon embedding resulted in the common ‘20-ntn ‘extracellular space and only partially triple-layered plasma membranes. The same tissue dehydrated with acetone and Vestopal embedded looked much like the equivalent 3-h postfixed material, i.e. it had apposed, triple-layered plasma membranes. When acetone dehydration and Epon embedding, or ethanol dehydration and Vestopal embedding were used with tissue of short postfixation time, the results were comparable. Apposed membranes interspersed with erratic amounts of extracellular space were common and plasma membranes tended to show an indistinct outer leaflet. Taken together the results indicate that the presence of apposed triple-layered plasma membranes is at least as valid as the common finding of a 20-nm space and partially tri-laminar membranes.
The effect of 6-hydroxydopamine on nerves in the rat upper urinary tract
Authors: Gosling J. A. , Dixon J. S.
Published: April 2021

The effect of a single large dose of 6-hydroxydopamine (6-OHDA) on adrenergic nerves in the rat upper urinary tract has been investigated by both light and electron microscopy. Reduction in the intensity of fluorescent catecholamine-containing nerves was seen 1–2 h after drug treatment. At 3–5 h, fluorescing nerves were absent from all preparations. Forty-eight hours after injection fine catecholamine-containing nerves could not be detected, although a few large nerves adjacent to vessels and in the adventitia of the upper urinary tract were associated with increased fluorescence. Using the electron microscope, axons related to smooth muscle were seen to be swollen and contained aggregations of small (50 nm diameter) granulated vesicles and electron-dense material 3–6 h after drug treatment. Other axons contained damaged mitochondria and were devoid of neurofilaments and neurotubules. Normal axons were also seen, some of which contained accumulations of small (50 nm diameter) agranular vesicles and occasional large (100 nm diameter) granulated vesicles. In the submucosa, damaged axons were observed lying close to, or occasionally running between, basal epithelial cells. Axons containing electron-dense clumps of synaptic vesicles were not observed in this situation. Normal axons containing accumulations of small agranular vesicles were identified, some of which were closely related to damaged axon profiles. Forty-eight hours after the drug treatment, some axons related to smooth muscle cells contained accumulations of electron-dense material. All other axons in the wall of the upper urinary tract appeared normal. These results are discussed in relation to the distribution of various types of autonomic nerve in the upper urinary tract.
Cellular junctions in the tunicate heart
Authors: Lorber V. , Rayns D. G.
Published: April 2021

The cell membranes of the tunicate heart exhibit an abundance of macular close junctions (also referred to as gap junctions or nexuses), distributed more or less randomly over the intercellular surfaces, and a system of zonal tight junctions limited to the cell borders adjacent to the epicardial surface. Three-dimensional images of these structures are displayed by the freeze-etch technique. In sectioned tissue, lanthanum appears to be excluded from the tight junctions, but is present in the close junctions. The presence of a primitive intercalated disk is suggested.
Cell electrophoresis of the cellular slime mould Dictyostelium Discoideum: II. relevance of the changes in cell surface charge density to cell aggregation and morphogenesis
Authors: Lee K.-C.
Published: April 2021

The reduction in the cell surface charge density of Dictyostelium discoideum during differentiation has been studied by the technique of cell electrophoresis. It was abolished under conditions in which cell aggregation was inhibited (e.g. low temperature, the presence of actinomycin D or cycloheximide). In the presence of nutrients incapable of supporting growth, cell aggregation occurred without a reduction in surface charge density. Cell adhesion in these aggregates was impaired, and a reduction in surface charge density appeared to be necessary for further development.

Brief treatment of exponential phase and aggregating cells with agents which disaggregate slugs failed to alter their electrophoretic mobilities. Low concentrations of magnesium chloride caused extensive agglutination, especially in aggregating cell suspensions, but little change in their electrophoretic mobilities. Magnesium chloride could agglutinate cells by association with cell surface components undetectable by cell electrophoresis. This, together with immunological evidence from other workers, supports the possibility of involvement of specific surface macromolecules in cellular slime mould aggregation. It was concluded that changes in surface charge density, though important for cell adhesion and morphogenesis, cannot account for all aspects of cell interactions in D. discoideum.
Characterization of dna-bound histone in the cells of the avian erythropoietic series
Authors: Appels R. , Wells J. R. E. , Williams A. F.
Published: April 2021

The distribution of DNA-bound histone from purified cell populations of the avian erythroid cell series was studied to examine the possible relationship between these molecules and the in vivo activity of cells. High-resolution polyacrylamide gel electrophoresis of histones indicated that the 3 main cell types, namely, erythrocytes (inactive in macromolecular synthesis), polychromatic erythrocytes (active in RNA and protein synthesis) and erythroblasts (dividing cells) all contained the same histone components. This result is contrary to previous reports that the f2c histone (characteristic of avian erythroid cells) was absent from erythroblasts; in addition it does not support the proposition that dividing cells contain a uniqueμ histone component. Quantitation of histone analyses showed that erythroblasts contain relatively less f2c histone than the non-dividing cells of the series and that there was a slight redistribution ofμ histone components between polychromatic and mature erythrocytes.
Spindle shape changes as an indicator of force production in crane-fly spermatocytes
Authors: Lafountain J. R.
Published: April 2021

Changes in spindle shape during the first and second meiotic divisions in living Nephrotoma suturalis spermatocytes have been studied and recorded cinematographically with phase-contrast, Nomarski differential interference, and polarization microscopy. With the Nomarski system 2 classes of discrete spindle filaments (new terminology) have been observed: continuous filaments that extend between the poles and appear to form a cage-like framework within which the chromosomes move, and kinetochore filaments which are attached to the chromosomes at their kinetochores and converge toward the poles. Spindle deformation occurs in a manner consistent with the hypothesis that whatever tensile forces are transmitted by the kinetochore filaments are balanced by compressive forces acting on the cage-like framework of continuous filaments causing them to become splayed. Just when these forces would be expected to reach a maximum, kinetochore filaments undergo a noticeable increase in contrast. It has not been determined whether this change represents a crowding of linear elements or addition of dry mass to a constant number of linear elements.
Structure and coiling of the stalk in the peritrich ciliates Vorticella and Carchesium
Authors: Amos W. B.
Published: April 2021

The stalk of Carchesium and Vorticella coils by the action of a contractile organelle. The organelle lies within a thread of cytoplasm which is encased in a complex extracellular tube. Study with the light microscope and the electron microscope suggests that the structure of the tube and the course of the organelle determine the form of the coiling. The contractile organelle contains a system of interconnected membranous tubules and the cytoplasm around it also contains membranous saccules. Both tubules and saccules extend along the length of the stalk.
Studies of chemotactic factor-induced polarity in human neutrophils : Lipid mobility, receptor distribution and the time-sequence of polarization
Authors: McKAY D. A. , Kusel J. R. , Wilkinson P. C.
Published: April 2021

Differences in membrane composition between the anterior and posterior poles of human blood neutrophils on exposure to chemoattractant were studied using a laser microscope, and the effects of attractant concentration and time on the ability of neutrophils to polarize were determined. The findings were as follows. (1) The fluorescein-labelled chemotactic hexapeptide fLeu-Norleu-Phe-Norleu-Tyr-Lys was asymmetrically distributed on polarized cells with the highest concentration at the anterior pole of the cell. (2) Differences in membrane lipid behaviour were studied by fluorescence recovery after photobleaching (FRAP) using the probe 5-N-(octadecanoyi)-aminofluorescein. Recovery curves suggested that the proportion of mobile lipid probe at the anterior pole of the cell was higher than at the posterior. However, no difference was found in the rate of recovery between the two poles. (3) Studies of the time-course of polarization were undertaken using neutrophils in suspension exposed to different isotropic concentrations of fMet-Leu-Phe (fMLP). At low concentrations (≤10-9M), the cells polarized immediately on exposure to the attractant. At a high concentration (10-7M) they assumed multipolar morphologies and polarized very poorly, suggesting that ligand binding was too rapid for the cells to form a dominant pole. At the optimal concentration, 10-8M fMLP, the cells assumed irregular, ruffled, morphologies after 30 s, but showed an increasing proportion of polarized forms over the next 30 min. These results suggest that, while cells exposed to low concentrations of attractant may respond by polarizing towards the site of a first signal from ligand, at optimal attractant concentrations a dominant pole is formed by integration of responses to ligand-binding at multiple sites. The polarization response is likely to be a stochastic process determined by chance events such as asymmetry or fluctuations in ligandreceptor binding.
Epidermal growth factor induces rapid reorganization of the actin microfilament system in human A431 cells
Authors: Rijken Philip J. , Hage Willem J. , Henegouwen Paul M. P. Van Bergen En , Verkleiji Arie J. , Boonstra Johannes
Published: April 2021

Double immunofluorescence microscopy reveals that epidermal growth factor (EGF) treatment of A43l cells results in more apparent co-localization of EGF receptor (EGFR) and actin fllaments, as compared to control cells. This indicates that EGF induces actin polymerization as well as additional association of the EGFR with similar sites on the membraneskeleton. We show that immunoprecipitation of the cytoskeleton-linked EGFR after fragmentation of the cytoskeleton results in specific co-precipitation of F-actin and a limited set of other unidentified proteins. Interestingly, EGF treatment of intact cells results in increased immunoprecipitation of cytoskeleton-associated EGFR as well as of F-actin, while actin does not co-precipitate with the non-ionic detergent-solubilized EGFR. These results demonstrate that the cytoskeleton-linked EGFR is associated with the actin microfilament system. EGF induces additional formation of protein complexes, containing the EGFR and F-actin and a limited set of other unidentified proteins. The increased co-pre-cipitation of F-actin is most likely related to EGF-induced actin polymerization, which is specifically associated with the apical cortical microfilament system, as demonstrated by confocal laser scanning microscopy and a phallicidin-binding assay.
Physarum plasmodia do contain cytoplasmic microtubules!
Authors: Salles-Passador Isabelle , Moisand André , Planques Viviane , Wright Michel
Published: April 2021

It has been claimed that the plasmodium of the myxomycete Physarum polycephaium constitutes a very unusual syncytium, devoid of cytoplasmic microtubules. In contrast, we have observed a cytoplasmic microtubule network, by both electron microscopy and immunofluorescence in standard synchronous plasmodia, either in semi-thin sections or in smears, and in thin plasmodia, used as a convenient model. Cytoplasmic microtubules could be seen after immunofluorescent staining with three different monospecific monoclonal anti-tubulin antibodies. The immunolabelling was strictly restricted to typical microtubules as shown by electron microscopy. These cytoplasmic microtubules were entirely and reversibly disassembled by cold treatment and by either of two microtubule poisons: methyl benzimidazole carbamate and griseofulvin. The microtubule network, present in all strains that have been studied, contains single microtubules and microtubule bundles composed of two to eight microtubules. Cytoplasmic microtubules form a dense and complex three-dimensional network, distinct from the microfilamentous domains and from the nuclei. The orientation of the microtubule network varies according to the plasmodial domain examined. Generally microtubules show no special orientation except in plasmodial veins where they are oriented parallel to the long axis of the veins. Differences between our observations and those of previous workers who failed to find cytoplasmic microtubules in plasmodia are discussed. We propose that they reflect difficulties of observation mainly due to the fluorescent background. In contrast with the previous view, the discovery of a microtubule cytoplasmic cytoskeleton in Physarum plasmodia raises several questions concerning its relationships with other cellular organelles and its dynamics during different cell cycle events.
Tubulin in sea urchin embryonic cilia: characterization of the membrane-periaxonemal matrix
Authors: Stephens R. E.
Published: April 2021

When the membranes of isolated embryonic cilia from three species of sea urchin are detergent-solubilized, the major proteins found are two equimolar polypeptides comigrating with tubulin subunits. Cross-reactivity with a variety of tubulin antibodies confirms their identity. Calmodulin and other calcium-binding proteins are prominent minor constituents of the extract. Removal of the solubilizing detergent by adsorption to polystyrene beads, followed by a freeze-thaw cycle, produces membrane leaflets and vesicles of uniform bouyant density. Such reconstituted membranes incorporate most of the tubulin and minor proteins but not calmodulin. Equivalent cross-reactivity with antibodies to acetylated or detyrosinated α-tubulin indicates that the tubulin derived from the mem-brane-periaxonemal matrix and axoneme are indistinguishable from each other in terms of these post-translational modifications but are distinct from the mainly unmodified tubulin of the embryonic cytoplasm. Pulse labeling with [3H]palmitate does not label either tubulin subunit but acylation does occur on a 190 ×103Mr membrane protein. Its specific activity is essentially the same whether label is applied to embryos with existing or regenerating cilia, suggesting rapid ciliary membrane protein exchange or physical turnover. Using pulse-chase labeling with [3H]leuc?ne during steady-state ciliary growth or induced regeneration in both normal and zinc-animalized embryos, the specific activity of the membrane-periaxonemal matrix-derived tubulin is initially higher than that of the axoneme but the degree of labeling equalizes in successive regenerations, consistent with derivation from a common pool. Many heavily labeled axonemal architectural proteins, such as tektin-A, are not reflected in the membrane-periaxonemal matrix fraction, suggesting that this fraction is not simply a pool of unassembled axonemal precursors.
Differential pathways of recruitment for centrosomal antigens to the mitotic poles during bipolar spindle formation
Authors: Maekawa Takami , Kuriyama Ryoko
Published: April 2021

As cells enter mitosis, centrosomes undergo many transformations and become associated with different molecules in a stage-specific manner. We have developed a protocol for immunofluorescence staining with four antibody probes that can help us to follow the interaction of centrosomal components during mitosis. The cells were first stained with a human autoimmune serum (5051); a monoclonal anti-phosphocentrosomal antibody (CHO3); and an antitubulin antibody. Localization of the antibodies was detected using rhodamine-, fluorescein- and AMCA-conjugated second antibodies, respectively. After photographing marked mitotic cells, coverslips were soaked with 0.2 M glycine–HCl at pH 1.0 for lh to release all antibodies bound to the structures. The same cells were re-stained with a human autoantibody (SP-H) specific for spindle poles and a fluor-escein-conjugated second antibody. This allowed us to compare the subcellular distribution of three kinds of centrosomal antigens in a single cell. Mitotic PtKi cells treated with either nocodazole or taxol included microtubule-containing cytoplasmic foci and parallel bundles of short microtubules at the cell periphery. All the centrosomal antibodies stained the same one or two dots corresponding to structures labeled by the tubulin antibody. CHO3 also revealed extra cytoplasmic foci, whereas the SP-H antigen was additionally localized at one end of the free microtubule bundles. As the microtubules reorganized into bipolar spindles during the recovery from drug treatment, the CHO3 and SP-H antigens coalesced into the spindle poles where the 5051 antigen was located, suggesting that centrosomal antigens become associated with spindle poles through very different recruitment pathways.
Experimental obliteration of the preprophase band alters the site of cell division, cell plate orientation and phragmoplast expansion in Adiantum protonemata
Authors: Mineyuki Yoshinobu , Murata Takashi , Wada Masamitsu
Published: April 2021

Division sites in higher plant cells are known to be determined before mitosis, and cell plates are precisely inserted into the predetermined division sites at the end of cytokinesis by unknown mechanisms. When apically growing protonemal cells of the fern, Adiantum capillus-veneris L., grown under red light are transferred to the dark, apical growth ceases and the protonemal cells then divide. However, this mitotic event can be influenced by subsequent exposure to light. If red-light pre-cultured protonemata are incubated in the dark and then transferred back to red light (after 28–36 h), apical growth resumes and the nuclei migrate toward the growing tips; interestingly, mitosis still occurs, although in an altered fashion. In the re-irradiated cells, timing of the premitotic nuclear positioning is delayed markedly and irregularly oriented cell plates are frequently observed. Re-irradiation with red light also causes an increase in cells without preprophase bands (PPBs) at prophase and the irregular expansion of the phragmoplast at late telophase, while early phragmoplast microtubule (MT) organization takes place normally. These data suggest the indirect involvement of PPBs in the guidance of phragmoplast expansion.
Topography of connexin32 in rat liver gap junctions : Evidence for an intramolecular disulphide linkage connecting the two extracellular peptide loops
Authors: Rahman Salman , Evans W. Howard
Published: April 2021

A range of anti-peptide antibodies directed towards selected amino acid sequences of connexin32 was prepared and characterised. The site-directed antibodies that identified connexin32 were used to study by immunolocalization and by proteolytic treatment of intact and split gap junctions the arrangement of the protein in the membrane. These studies reinforce models of connexin topography in which the polypeptide traverses the junctional membrane four times, with the amino and carboxyl termini cytoplas-mically located. The four transmembrane domains were shown to be linked by two extracellular loops with a single intracellular loop connecting the second and third transmembrane domains. Evidence is presented to show that the two extracellular domains of connexin32, which are important for intercellular adhesion and the insulated bridging of the extracellular space by channels allowing cellcell communication across the gap junction, are connected by disulphide bond(s). The studies lead to a more detailed two-dimensional model of con-nexin32 in the membrane, incorporating the favoured theoretical arrangement of disulphide bonds at the extracellular domain of connexin32.
Distribution of fibronectin and fibronectin-binding proteins, AGp110 and integrin α
5
β
1 during chemically induced hepatocarcinogenesis in adult rats
Authors: Stamatoglou Stamatis C. , Manson Margaret M. , Green Jonathan A. , Mayol Xavier , Hughes R. Colin
Published: April 2021

We have used single- and double-label immunocytochemistry to examine the distribution of AGpllO, integrin α5β1 and fibronectin in adult rat liver during carcinogenesis induced by aflatoxin B2 or diethyl-nitrosamine. In normal liver fibronectin and the fibronectin integrin receptor α5β1 are localized on all three domains of the parenchymal cell surface: sinusoidal, lateral and canalicular. In contrast, AGpllO, a non-integrin monomeric glycoprotein with fibronectin receptor properties, is confined to the bile canalicular (apical) plasma membrane of hepatocytes. Hepatocarcinogenesis induced by aflatoxin B2 causes altered cell foci to form in the parenchyma, followed by enlargement of these foci to form pre-neoplastic nodules and finally hepatocellular carcinomas of either poorly differentiated, trabecular or adenocarcinoma morphology. Expression of AGpllO decreased to a minimal level, at first selectively in altered cell foci, from the 9th week of treatment, and then indiscriminately in poorly differentiated carcinomas. The same lesions that were deficient in AGpllO also displayed a reduced level of fibronectin and 25βi, although the observed change in AGpllO demarcated altered foci and poorly differentiated tumour lesions more sharply, since expression of α5β1 and fibronectin, though substantially reduced, was still faintly apparent on the cell surface. Small acinar structures, observed in late hyperplastic nodules and in trabecular carcino mas, exhibited even, pericellular staining of fibronectin and α5β1 including prominent staining of the lumen area, whereas staining of AGpllO appeared to be confined to the lumen. In larger ducts of overt adenocarcinomas, fibronectin and α5β1 were distributed along the basal surface of the epithelium and AGpllO on the apical domain. Tumours induced by diethylnitrosamine and promoted with ethinyl estradiol displayed similar histology and staining patterns for all three proteins as that described for aflatoxin B2. Finally, comparisons between AGpllO and cytokeratin 19, a selective tumour marker, indicated that whereas loss of AGpllO occurs in poorly differentiated lesions and tumours, expression of cytokeratin 19 is associated with acinar and glandular structures found in late hyperplasia and with trabecular and pseudoglandular tumours. The results indicate that loss of differentiation in either hyperplastic or neoplastic lesions correlates with reduced expression of fibronectin and of its receptors α5β1 and AGpllO. On the basis of morphological similarities and staining patterns in the pre-neoplastic and neoplastic state, we deduce that hepatocellular carcinomas derive from differentiated hepatocytes.
Characterisation of potential adhesins of the bacterium Pasteuria penetrans, and of putative receptors on the cuticle of Meloidogyne incognita, a nematode host
Authors: Lay A. Persidis, J. G. , Manousis T. , Bishop A. H. , Ellar D. J.
Published: April 2021

Pasteuria penetrans spores were fragmented by glass bead vortexing, producing exosporiai membranes and spore fragments, which consisted of fibre bundles. Both exosporia and spore fragments are capable of host-specific attachment to the cuticle of Meloidogyne incognita, a root-knot nematode host. Putative M. incognita receptors appear to be soluble in β-mercaptoethanol (BME) but not SDS, and are also sensitive to tryptic digestion and deglycosylation by endoglycosidase F. Polyclonal antibodies against intact spores and spore fragments inhibited attachment up to 25% and Fab’ fragments of antispore antibodies produced 100% inhibition. The antibodies, however, did not show preferential staining of particular spore structures in thin section immunolabelling studies. Exposure of Pasteuria penetrans spores to HC1 or urea-SDS-dithiothreitol renders them incapable of attachment to their host juveniles and extensively disrupts fibres that surround the spore core. Protein extracts from spore fragments or from exosporiai membranes are identical, and urea-BME extracts from either structure, but not SDS extracts, can inhibit the attachment of spores to juveniles by 60-80%. An inhibitory BME extract from spore fragments was analysed by anion-exchange chromatography and adsorption onto host cuticle followed by immunoblotting. It appeared to contain six potential spore adhesins of approximate Mr 24-29, 38-47, 59, 89, 126, and 190 (×IO3). Lectin affinity blotting with wheat germ agglutinin and concanavalin A showed that all of these proteins bear terminal N-acetylglucosamine residues and the 38–47 kDa band also bears terminal Glc/Man residues. A BME extract from strain Pp 1 inhibited the attachment of strain PNG to M. incognita and vice versa and the PNG strain had a greater amount of two proteins of 98 and 126 kDa than the Pp 1 extract.
Hormonal regulation of spermatid binding
Authors: Cameron Don F. , Muffly Karl E.
Published: April 2021

A Sertoli-spermatid coculture model is described in which a large percentage (>76%) of round spermatids remain viable for 48 h and bind to Sertoli cells. The effects of follicle-stimulating hormone (FSH) and testosterone on spermatid binding (expressed as the spermatid density; SD- the number of spermatids per unit area of Sertoli cell cytoplasm), ultrastructure of the Sertoli-spermatid junctional complex, and distribution in the Sertoli cell of junction-related F-actin and vinculin are described. Following 48 h of incubation, neither FSH alone nor testosterone alone affected spermatid binding to Sertoli cells beyond that observed in control cocultures. However, the combination of FSH and testosterone (FSH+testosterone) resulted in a significant increase in the density of spermatids bound to Sertoli cells. Junction-related structure of the Sertoli cell cytoskeleton between the Sertoli cell and the pre-step 8 spermatid was different than that observed between the Sertoli cell and the post-step 8 spermatid. The junction-related cytoskeletal modification of the Sertoli cell (JCMS) in the latter was similar in appearance to the well-described ‘Sertoli ectoplasmic specialization* observed adjacent to post-step 8 spermatids in vivo. FSH+testosterone and FSH alone, but not testosterone alone, resulted in the peripheral distribution of actin and vinculin, which otherwise remained in stress fiber-like structures throughout the Sertoli cell. Results show that maximal spermatid binding to Sertoli cells in vitro requires FSH+testosterone and is associated with the peripheral distribution of actin and vinculin.