bluuebunny/crossref_metadata_2025 huggingface dataset:
Adhesive properties of mdck cell membranes: possible role in epithelial histogenesis
Authors: Whitesell Luke , Edwardson J. Michael , Cuthbert Alan W.
Published: April 2021

MDCK cells in culture form a functional transporting epithelium. Apical surfaces of MDCK monolayers are not adhesive to free MDCK cells that fail to grow attached to existing monolayers. In contrast, when plated at high density (10 6 cm -1) MDCK cells form multilayered structures in which only the outermost layer shows a typical apical surface (microvilli, tight junctions, etc.). In the lower layers cell-cell contacts (desmosomes, etc.) are made, suggesting that the basolateral surfaces remain adhesive.

Using a novel, quantitative, heterotypic cell adhesion reaction with aged human red cells, we have measured the adhesive properties of the apical surfaces of MDCK cells in a variety of conditions. It is found that the adhesion of red cells is dependent on cell density in the monolayer and falls to a low value at confluence. The reaction is considerably stronger at 6 °C compared to 22 °C (the temperature of most studies), while at 37 °C the reaction is expressed less strongly. MDCK cells with adherent red cells fail to divide, suggesting that the cell surface receptors involved might mediate growth inhibition by a similar homotypic reaction at confluence. The expression of the adhesive property is associated with cell division. This was shown by inducing synchrony in cultures either by refeeding starved cultures or by removal of a thymidine ‘block’. Peaks in adhesiveness were related to peaks in thymidine incorporation in synchronized cultures.

Because of the precise quantitative way in which adhesion can be measured it is considered that this is an ideal system in which to identify the membrane components responsible for the reaction. Furthermore, it may prove possible to identify signals that arise as an immediate consequence of the adhesion reaction.
Cytoskeletal and transmembrane interactions in the expression of tumourigenicity in human cell hybrids
Authors: Der Channing J. , Ash J. F. , Stanbridge Eric J.
Published: April 2021

The cytoplasmic cytoskeletal organization was examined by immunofluorescence analysis in a series of paired non-tumourigenic and tumourigenic HeLa/fibroblast hybrids. The microfilament and microtubule complexity in tumourigenic hybrids was compared to that of the corresponding transformed, non-tumourigenic hybrids from which they were derived. Since both the non-tumourigenic and tumourigenic hybrids displayed well-organized microtubules, the disruption of these cytoskeletal elements is not required for transformation or tumourigenicity. In contrast, the microfilament organization was drastically altered in association with the re-expression of tumourigenicity. However, treatment of these tumourigenic segregant lines with sodium butyrate or dexamethasone resulted in a more flattened morphology and the appearance of well-developed microfilament bundles. This indicated that microfilament complexity was related to cellular morphology and not to tumourigenic potential. Finally, results of double immunofluorescence analyses for microfilaments and fibronectin staining in these hybrids agree with the existence of a transmembrane association between these 2 systems. In both normal fibroblasts and non-tumourigenic hybrids a close, but not identical, distribution was seen for surface fibronectin and cytoplasmic microfilaments. In the tumourigenic hybrids the lack of a well-organized miciofilament system was associated with a much reduced stitched-like pattern for fibronectin. The antibody-induced redistribution of surface β2-microglobulin reflected this loss of a transmembrane influence on the organization of surface components. Unlike the non-tumourigenic hybrids, the tumourigenic segregants possessed an altered extracellular matrix, rounded morphology, and reduced adhesion capabilities. The corresponding reduction in microfilament organization in these hybrids suggests that there is a close interrelationship between these cellular parameters. The exact contribution of these alterations to tumourigenic potential is unclear but the apparent loss of the transmembrane interaction with the cell surface organization may well represent a crucial alteration central to in vivo growth potential.
The abnormal morphology of polyoma-transformed baby hamster kidney cells is due to a failure to respond to 70k spreading factor
Authors: Knox Peter , Griffiths Sheila
Published: April 2021

The typical elongated bipolar morphology of baby hamster kidney (BHK) cells is not shown by polyoma-transformed BHK (Py-BHK) cells. Instead, the transformed line adheres poorly to tissue-culture plastic and cells have a more rounded morphology than the parent line. Plasma fibronectin is known to mediate the spreading of BHK cells, but when human serum is subjected to Sephacryl S-300 chromatography two peaks of spreading activity are eluted; the first is fibronectin and the second, which is quantitatively more significant, is a 70 K protein that is not related to fibronectin and stimulates spreading by a different mechanism. Py-BHK cells spread well in low concentrations of purified fibronectin but will not spread well in levels of serum that contain similar of greater concentrations of fibronectin. This is because fibronectin-mediated spreading of both BHK and Py-BHK cells occurs only in the presence of low concentrations of other proteins; albumin and other serum proteins inhibit fibronectin-mediated spreading. BHK cells spread under routine culture conditions in response to the 70 K factor rather than fibronectin. The altered morphology that results from viral transformation is due to a failure of the cells to respond to the 70 K spreading factor.
Biliary secretion of sodium fluorescein in primary monolayer cultures of adult rat hepatocytes and its stimulation by nicotinamide
Authors: Gebhardt Rolf , Jung Wilhelm
Published: April 2021

Addition of sodium fluorescein to primary cultures of rat hepatocytes resulted in a rapid uptake of the dye by the hepatocytes and a subsequent accumulation in bile canaliculi-like structures. A similar distribution was obtained with fluorescein diacetate. Concentrations of Na-fluorescein accumulating within canaliculi varied over a wide range, often far exceeding that used in the medium. Ouabain strongly blocked cellular uptake, thus also impairing secretion of Na-fluorescein, whereas colchicine affected neither process. Taurolithocholate had virtually no influence on uptake, but markedly reduced the number of fluorescent canaliculi. Furthermore, fluorescent canaliculi could be discharged by addition of 1 M-sucrose in Hank’s buffer, without affecting viability of the cultured cells.

The percentage of canicular structures accumulating high amounts of Na-fluorescein markedly increased during cultivation for 7 days, concomitant with the progressive development of originally small and sporadic canaliculi into an anastomosing network of slender channels. This canalicular proliferation was strikingly reinforced by 20 mM-nicotinamide, resulting in an impressive network of canaliculi within 2–3 days. Nicotinamide also supported the secretion of Na-fluorescein, which could be stimulated further by addition of dehydrocholate.

These results suggest that cultured hepatocytes are able to re-create a functional biliary polarity at least with respect to the biliary secretion of Na-fluorescein.
In vitro proliferation and lifespan of bovine aorta endothelial cells: Response to conditioned media
Authors: W. Lincoln David , Whitney Ronald G. , Smith James R.
Published: April 2021

The effect of conditioned medium on the growth rate of bovine aorta endothelial cells (CSC311) was examined. Media conditioned by several cell types (both immortal and normal) was found to increase the growth rate of CSC311 cells-The growth rate of CSC311 was increased even when the medium was conditioned by other CSC311 cells. The rate of growth of unstimulated CSC311 cells is 0·43 population doublings (p.d./day. Conditioned medium from several cell types gave a maximum stimulated growth rate of 1·3 p.d./day. In addition, we present evidence for an increase in the degree of stimulation by the growth-promoting activity found in conditioned medium with increasing in vitro age of CSC311 cells.
Mechanism of fc-mediated interaction of eosinophils with immobilized immune complexes: I. effects of inhibitors and activators of eosinophil function
Authors: Oliver Rhonda C. , Glauert Audrey M. , Thorne Kareen J. I.
Published: April 2021

A protein of apparent molecular weight 55000, designated protein 3, becomes newly detectable on the eosinophil surface as a specific consequence of interaction with antigen-antibody complexes immobilized in agar layers. The effect of various agents upon this interaction has been determined by monitoring the appearance of this protein by lactoperoxidase-catalysed iodination. Other parameters that have been measured include: the attachment of eosinophils to the agar layers and their subsequent degranulation, as measured by the release of granule peroxidase, and the degree of spreading of the eosinophils, as assessed by electron microscopy.

Attachment of eosinophils to antibody-coated layers is inhibited by heat-aggregated immunoglobulin G (IgG), suggesting that this attachment is mediated via eosinophil Fc receptors. In addition, agents, such as the eosinophil chemotactic factor Ala-Gly-Ser-Glu, that enhance the expression of Fc receptors also enhance the appearance of protein 3, while agents, such as hydrocortisone, that inhibit the expression of Fc receptors reduce its appearance. It is concluded that the appearance of protein 3 parallels the expression of Fc receptors.

Attempts to block the Fc region of the bound antibody with staphylococcal protein A were not successful. These experiments indicated that the Fc region of bound IgG has different binding sites for protein A and for the Fc receptor.

The correlation between the appearance of protein 3 and subsequent degranulation of the eosinophils was confirmed by the use of agents, such as cytochalasin D and levamisole, that enhance both the appearance of protein 3 and degranulation. Conversely, hydrocortisone reduces the appearance of protein 3 and inhibits degranulation.

Protein 3 does not appear when eosinophils adhere to agar layers coated with concanavalin A instead of antibody and the eosinophils do not degranulate. Addition of the calcium ionophore A23187, while causing the release of granule peroxidase, does not elicit the appearance of protein 3. These observations provided additional evidence that the appearance of protein 3 is a specific consequence of the interaction of eosinophils with antibody-coated surfaces.

The fact that protein 3 appears at the eosinophil surface as a direct consequence of the interaction with antibody suggests that this protein is closely associated with the eosinophil Fc receptor. The enhancement of the appearance of protein 3 in the presence of cytochalasin D indicates that the movement and reorientation of both this protein and the Fc receptor are constrained by association with cytoplasmic microfilaments.
The mechanism of fc-mediated interaction of eosinophils with immobilized immune complexes ii. identification of two membrane proteins, modified by the interaction
Authors: Thorne Kareen J. I. , Free Jill , Franks David , Oliver Rhonda C.
Published: April 2021

Human peripheral blood eosinophils attach to and flatten down onto antibody-coated surfaces and subsequently degranulate. An antibody-coated surface was prepared by treating a layer of agar, containing tetanus toxoid antigen and eosinophil chemotactic factor (EOF), with human anti-tetanus immunoglobin. Changes in eosinophil surface proteins during attachment to the antibody-coated agar layer were detected by lactoperoxidase catalysed iodination. Purified eosinophils were pre-treated with unlabelled iodide, lactoperoxidase and hydrogen peroxide to block pre-existing accessible tyrosine residues on the cell surface. They were then allowed to interact with the agar layer, and subsequently treated with lactoperoxidase and luI-labelled iodide to label newly accessible surface proteins. Separation of the radioactive proteins by sodium dodecyl sulphate/polyacrylamide gel electrophoresis revealed that, while incubation of the cells in suspension restored the major proteins to the cell surface, interaction with the antibody-coated agar layer caused the appearance of additional proteins of apparent molecular weight 55K, 30K, 28K and 18K.

The 55K, 28K and 18K proteins were greatly reduced when antibody was absent, but the 55K protein was distinguishable from immunoglobulin G (IgG) heavy chain, since it could be detected in low amounts even in the absence of antibody. It was found in purified plasma membranes and it could be separated from IgG heavy chain by iso-electric focusing. The possibility is discussed that this protein is either linked to the receptor for the Fc portion of IgG, or that it is itself the receptor.

The 18K protein required both antibody and ECF for maximum expression, but was seen in limited amounts with ECF alone. Possibly it is concerned with an ECF-mediated recognition of IgG. Unlike the 55K protein, it binds concanavalin A.

Plasma membranes were prepared from eosinophils by lysis in borate, followed by purification on a glass-bead column. Both the 55K and the 18K proteins were found to be major components of the eosinophil membrane.
An ultrastructural study of the interaction in vitro between Trypanosoma theileri and bovine leucocytes
Authors: Townsend Jackie , Duffus W. P. H. , Glauert Audrey M
Published: April 2021

Bovine neutrophils, eosinophils and macrophages obtained from the mammary gland were cytotoxic to Trypanosoma theileri epimastigotes in vitro in the presence of specific antibody. A detailed ultrastructural study revealed that all types of effector cell phagocytosed T. theileri despite the relatively large size of this parasite (10–130μm).

Phagocytosis proceeded in the classical manner by envelopment of the trypanosome by long pseudopodia. With eosinophils and macrophages structural damage of the parasite was only observed following ingestion. There was evidence of inter-granule fusion in eosinophils prior to the discharge of granule contents into the parasitophorous vacuole; following degradation of the parasite in eosinophils some phagolysosomes opened to the exterior and released what appeared to be trypanosome debris. While eosinophils required less than 30 min to initiate serious damage to the parasite, macrophages required in excess of 2 h to produce similar damage. Conversely, neutrophils caused severe damage within 5 min.

An important feature of neutrophil-mediated cytotoxicity was the extensive structural damage to the parasite that was observed before closure of the phagosome. Initially this damage was observed at the end of the trypanosome that was in contact with the neutrophil. This finding suggests that neutrophil-mediated killing of T. theileri is a combination of both extracellular and intracellular mechanisms.
Structure and chemical composition of insoluble filamentous components of sperm flagellar microtubules
Authors: Linck R. W. , Langevin G. L.
Published: April 2021

By progressive solvent extraction, we have obtained a series of subfragments of flagellar microtubules. Mild treatment gives rise to ribbons that contain longitudinally arranged protofilaments. Further extraction leaves a distinctive residue containing thinner ribbons, of three and eventually two protofilaments. Finally, filaments 2–3 nm in diameter and fibrous ribbons apparently containing 6 or more 2 nm subfibrils are found. This latter solvent-resistant material is consistently enriched in a characteristic set of polypeptides, which are found in flagella of several different species, including echinoderms and a mollusc. These polypeptides appear different from α- and β-tubulin on the basis of their solubilities, isoelectric points and electrophoretic mobilities in sodium dodecyl sulphate/polyacrylamide gels; these conclusions are reinforced by peptide mapping after limited proteolytic digestion, although the latter method reveals certain similarities between these unique flagellar proteins, tubulin, chicken gizzard desmin and rabbit actin. A remarkable feature of the protein in the final fraction is the high a-helical content: 71 % as measured by circular dichroism. We consider the possible origins of these filaments in the microtubule, in particular the possibility that microtubule protofilaments are heterogeneous in protein composition, and we discuss some of the implications of our findings.
Gene expression in Acetabularia: I. Calibration of wheat germ cell-free translation system proteins as internal references for two-dimensional electrophoresis
Authors: Shoeman Robert L. , Schweiger Hans-Georg
Published: April 2021

Modification of existing two-dimensional techniques enables isoelectric focusing and sodium dodecyl sulphate polyacrylamide gel electrophoresis of complex mixtures of proteins to be completed within 8 h. The method was optimized to separate the protein components of a wheat germ cell-free translation system, providing a statistically proven resolution better than 0 03 of a pH unit for the isoelectric point and 1000 for Mr. Fourteen of the more than 300 proteins separated were characterized with respect to Mr and isoelectric point relative to standard proteins under the same conditions. Stained wheat germ proteins thus serve as internal standards for analysis of in vitro translation products.
Patterns of protein synthesis during the cell cycle of the fission yeast Schizosaccharomyces pombe
Authors: Creanor J. , Mitchison J. M. , Williams D. A.
Published: April 2021

The rate of protein synthesis through the cell cycle of Schizosaccharomyces pombe has been determined from the incorporation of pulses of [3H]tryptophan in synchronous cultures prepared by selection in an elutriating rotor. This selection procedure caused minimal perturbations as judged by asynchronous control cultures, which had also been put through the rotor. The rate of synthesis showed a periodic pattern rather than a smooth exponential increase. There was a sharp increase in the rate at an ‘acceleration point’ at about 0-9 of the cycle. Model-fitting by a novel procedure suggests that the average single cell has an increasing rate of protein synthesis for the first 60% of the cycle and a constant rate for the remaining 40%.

The same pattern was shown in less extensive experiments with [3H]leucine and [3H]phenyl- alanine. It was also shown in a series of size mutants, which indicates that the pattern is not size-related, in contrast to earlier work on the rates of synthesis of messenger RNA. However, one large mutant (cdc z.M35r20) had a significantly earlier acceleration point.

Care was taken to justify the assumption that the rate of incorporation of tryptophan was a valid measure of the rate of protein synthesis. A tryptophan auxotroph was used to eliminate the problem of endogenous supply and the size of the metabolic pool was measured through the; cycle. This pool did not show cell-cycle related fluctuations. An operational model of the pools is presented.
Calcium-sequestering vesicles and contractile flagellar roots
Authors: Salisbury J. L.
Published: April 2021

The intracellular localization of calcium during contraction of striated flagellar roots of the green alga Tetrasehnis subcordiformis was investigated using calcium pyroantimonate and calcium oxalate cytochemistry and energy-dispersive X-ray microanalysis.

Vesicles (0·25-0·50 μm diameter) located in the anterior-most region of the cell are calcium-sequestering organelles. These vesicles exhibit a consistent and reproducible localization of electron-opaque pyroantimonate and oxalate salts of calcium. Striated contractile flagellar roots (CFR) show a periodic pattern of calcium antimonate (Ca-Sb) deposits following ‘calcium shock’ stimulation of contraction of this organelle. When CFRs are in a fully extended state they do not show Ca-Sb deposits. Deposits of Ca-Sb precipitate are present throughout the cytosol when CFRs are stimulated to contract.

The implications of these findings with respect to the regulation of intracellular levels of ‘free ‘Ca2+ and to the mechanism of CFR contraction are discussed.
Glycoconjugates from high endothelial cells: I. Partial characterization of a sulphated glycoconjugate from the high endothelial cells of rat lymph nodes
Authors: Andrews P. , Milsom D. W. , Stoddart R. W.
Published: April 2021

Rat lymph nodes secrete a sulphated macromolecule. This molecule was isolated by ion-exchange chromatography and partially characterized by gel-permeation chromatography, before and after chemical and enzymic modification, and electrophoresis on polyacrylamide gels, paper and glassfibre paper. The results obtained suggest that the label was not incorporated into a proteoglycan but entered a glycolipid that is non-covalently linked to a glycoprotein. A possible role for this complex is discussed.
Comparative study of extracellular fibrils on the ectodermal layer in gastrulae of five amphibian species
Authors: Nakatsuji Norio , Johnson Kurt E.
Published: April 2021

Previous studies have shown the presence of a network of extracellular fibrils on the inner surface of the ectodermal layer of Ambystoma maculatum gastrulae. The alignment of the network along the blastopore-animal pole axis has suggested that the network of fibrils guides the migrating mesodermal cells in gastrulae by contact guidance. We have also shown that these fibrils can be deposited on substrata by explanted embryonic fragments and that substrata conditioned in this manner support directed cell migration. In this study, we found that the appearance of the fibrils in the embryos coincides with the start of cell migration towards the animal pole. Gastrulae of three urodele species examined (A. maculatum, A. mexicanum and Cynops pyrrhogaster) have similar dense networks of fibrils. Xenopus laevis gastrulae also have similar fibrils but fewer fibrils compared to urodele embryos. Rana pipiens gastrulae have very few extracellular fibrils. The scarcity of the fibrils in anuran species may be related to the differences in arrangement of mesodermal cells during migration.
Ultrastructural changes in keratinizing epithelium following trypsinization, epidermal detachment and apposition to mesenchymes
Authors: Jensen H. M. , Mottet N. K.
Published: April 2021

The cellular effects of trypsinization, a commonly used method for separating epithelial from mesenchymal tissues, were examined with the electron microscope. Specimens were fixed after each step of the trypsinization process in 1 % OsO4 in double strength Tyrode solution and after 1–7 days of culture following reapposition of the epithelium to its own mesenchyme or collagen-rich 12-day embryonic quadriceps tendon. Controls consisted of specimens identically prepared from limb bud skin derived from the same site from s-day normal embryos to hatching. A second series of controls were 5-day skin explants cultured for 1-7 days in vitro following identical preparation except for the omission of trypsin.

Trypsin caused transient cytoplasmic protrusions (‘blebs’) on the deep aspect of the basal cells, cellular shrinkage and increase in lysosomes. The basal lamina was disrupted overlying the blebs and remained attached to the basal cells only between the blebs. Continuity of the basal lamina was restored within 24 h after the onset of cultunng and developed precociously thereafter, regardless of whether it was reapposed to its own collagen-poor mesenchyme or the collagen-rich quadriceps tendon. In the latter, focal excessive basal lamina formation was observed. Epithelial cell differentiation proceeded equally precociously in trypsinized and non-trypsinized cultures. Disruption of the basal lamina by trypsinization did not produce detectable alteration in the synthesis of tonofilaments, desmosomes, granular reticulum or corpuscula cnbriformia. Keratohyaline bodies were not found in any of the cultures and keratinized cells did not develop.
Natural variability in the length of thin and thick filaments in single fibres from a crab, Portunus Depurator
Authors: Franzini-Armstrong Clara
Published: April 2021

The carpopodite flexor of the walking legs of the crab Portunus depurator contains fibres belonging to 3 groups. These are characterized by differences in the cross-striation spacing. Fibres having sarcomeres of approximately 4, 5 and 7 μm are here called short, medium and long sarcomere types, respectively.

Within individual fibres belonging to any of the groups the length of the A band is not constant. Up to 25 % length differences have been measured in A bands belonging even to the same fibril.

The bridge-free regions of the thick filaments are not always in the centre, so that the filaments are often asymmetric. Analogally, the L line, resulting from the alignment of the bridge-free regions of the thick filaments, may be asymmetrically placed in the Z band. The length of the bridge-free region in crab thick filaments is 60 nm, while the corresponding region in vertebrate thick filaments is 120 nm. This is discussed in terms of a possible model of the filament.

The length of the thin filaments is proportional to that of the thick filaments in the corresponding portion of the sarcomere. When two A bands of different length occur in adjacent positions along the fibril, the Z line is not a centre of symmetry.

The ratio of thin to thick filament number is variable in individual fibrils. In general, the ratio is higher in the medium sarcomere type fibres than in the short sarcomere type.

Stretched and shorter portions of single fibres of the medium type have been examined and the A-band length populations compared. From such a study it can be deduced that passive length changes occur in crab fibres by sliding of thin and thick filaments.
Phase equilibria of cytoplasmic actin of cultured epithelial (BHK) cells
Authors: Koffer Anna , Gratzer W. B. , Clarke G. D. , Hales Anne
Published: April 2021

The actin in cold lysates of cultured baby hamster kidney cells was predominantly monomeric, as judged by the DNase inhibition assay. Most of the filamentous actin attached to the membranes could be dissociated by washing with low ionic strength buffer, but a minor part was tightly associated in complexes with other proteins and could not be liberated for assay by DNase after exposure to a depolymerizing medium.

The monomeric actin in the cell extract must be sequestered as a complex with a profilin-like protein, since its concentration was greatly in excess of the critical concentration for polymerization. On warming, rapid polymerization ensued, with loss of most of the monomeric actin. A separate cross-linking process could be observed by light-scattering measurements. A gel was formed, that contained besides actin substantial amounts of constituents identified as filamin, myosin, a-actinin, a gelsolin-like protein and other species of apparent subunit molecular weights 58 000 and 300 000, presumed to derive from intermediate filaments. Some low molecular weight protein subunits, including tropomyosin, were also present. The gelation was not accompanied by a significant degree of phosphorylation of any of the actin-associated proteins, with the exception of the minor species of 300000 molecular weight, the phosphorylation of which was cAMP-dependent.

In the presence of concentrations of free calcium in the sub-micromolar range, the gel initially formed contracted and gradually dispersed. At higher concentrations of free calcium a loose turbid gel formed, but the bulk of the actin was found in the supernatant after high-speed centrifugation. Using a fluorescent actin derivative as a tracer, the quantum yield of which increases by nearly two orders of magnitude when it is incorporated into a polymer, it was established that the actin in the supernatant was not monomeric, but rather in the form of very short filaments. It was shown that scission of filaments accompanied the contraction of the cytoplasmic gel at the lower concentrations of calcium. The short filaments were highly effective in nucleating the polymerization of added G-actin and were evidently rapidly dissociated by DNase, since they reacted as monomers in the DNase inhibition assay.

The gel obtained in the absence of calcium was revealed by electron microscopy to consist of clusters of filaments, many of which show a highly regular lateral spacing of about 7·0 nm.

The physical basis for the temperature- and calcium-dependent phase equilibrium observed in this system are analysed in semi-quantitative terms.
Substrata formed by combinations of extracellular matrix components alter neural crest cell motility in vitro
Authors: Erickson C. A. , Turley E. A.
Published: April 2021

Extracellular matrix components such as collagen, fibronectin and sulphated glycosaminoglycans can act as substrata that promote neural crest motility in vitro, in the absence of serum. The cells appear to be less adhesive and move more randomly on collagen or chondroitin sulphate substrata than on fibronectin substrata. Cells do not spread or become motile on plastic dishes to which hyaluronate has been bound, presumably owing to weak adhesion to this surface. Hyaluronate added to the medium alone has little effect on cell motility. When combinations of matrix molecules are used as substrata, however, the presence of fibronectin increases spreading, directional persistence of cell motility and speed of movement above that observed on collagen alone. When added to fibronectin, chondroitin sulphate appears to reduce adhesions slightly, since the cells are more rounded. Hyaluronate added in the medium significantly reduces the extent, speed and directionality of movement on fibronectin substrata. The presence of collagen in combination with fibronectin plus glycosaminoglycans does not have a noticeable effect on cell motile behaviour, beyond that observed with fibronectin alone. The effects of combinations of matrix compounds on neural crest cell motility are thus predictable, and can be explained in terms of the known adhesive properties and reported binding interactions of these molecules. These studies in vitro are compared with neural crest cell motility in vivo.
Studies of nuclear and cytoplasmic behaviour during the five mitotic cycles that precede gastrulation in Drosophila embryogenesis
Authors: Foe Victoria E. , Alberts Bruce M.
Published: April 2021

Using differential interference contrast optics, combined with cinematography, we have studied the morphological changes that the living, syncytial embryo undergoes from stage 10 through 14 of Drosophila embryogenesis, that is just prior to and during formation of the cellular blastoderm. We have supplemented these studies with data collected from fixed, stained, whole embryos. The following information has been obtained.

The average duration of nuclear cycles 10, 11, 12 and 13 is about 9, 10, 12 and 21 min, respectively (25 °C). In these four cycles, the duration of that portion of the mitotic period that lacks a discrete nuclear envelope is 3, 3, 3 and 5 min, respectively. The length of nuclear cycle 14 varies in a positionspecific manner throughout the embryo, the shortest cycles being of 65 min duration.

During nuclear cycles 10 through 13, it is commonly observed in living embryos that the syncytial blastoderm nuclei enter (and leave) mitosis in one of two waves that originate nearly simultaneously from the opposite anterior and posterior poles of the embryo, and terminate in its midregion. From our preparations of quick-frozen embryos, we estimate that these mitotic waves take on average about half a minute to travel over the embryonic surface from pole to equator.

The yolk nuclei, which remain in the core of the embryo when the rest of the nuclei migrate to the periphery, divide in synchrony with the migrating nuclei at nuclear cycles 8 and 9, and just after the now peripherally located nuclei at nuclear cycle 10. After cycle 10, these yolk nuclei cease dividing and become polyploid.

The syncytial embryo has at least three distinct levels of cytoskeletal organization: structured domains of cytoplasm are organized around each blastoderm nucleus; radially directed tracks orient colchicine-sensitive saltatory transport throughout the peripheral cytoplasm; and a long-range organization of the core of the embryo makes possible coherent movements of the large inner yolk mass in concert with each nuclear cycle. This highly organized cytoplasm may be involved in providing positional information for the important process of nuclear determination that is known to occur during these stages.
Reversible disruption of cultured endothelial monolayers by sulphated fucans
Authors: Glabe Charles G. , Yednock Ted , Rosen Steven D.
Published: April 2021

We have examined the effects of a variety of polysaccharides and glycoconjugates on the organization and morphology of cultured AI4CL-I endothelial monolayers. The sulphated fucose-containing polysaccharides, fucoidin and sea-urchin egg jelly fucan, induce a dramatic disruption of the organization of the monolayers, characterized by the retraction of adjacent borders of cells exposing areas of the subendothelial matrix. This effect, which occurs at a fucoidin concentration of 10 μg/rnl, is rapidly reversible after the fucoidin-containing medium is removed. Within 1 h after replacement with fresh medium many cell contacts are re-established; within 20h the fucoidin-treated monolayers closely resemble the untreated controls. The effect of the sulphated fucose-containing polysaccharides is specific. Of a wide variety of sulphated polysaccharides and glycoconjugates tested, only fucoidin and the egg jelly fucan produce a detectable alteration in the morphology of cultured endothelial monolayers. The endothelial monolayer has specific binding sites for fucoidin. The binding of fucoidin is saturable and a maximum of 4·5 × 105 molecules of fucoidin are bound per cell with an apparent affinity of 2·3 × 10−7M. A significant proportion (26%) of the total monolayer-associated fucoidin is apparently internalized by the endothelial cells after incubation with fucoidin for 1 h at 37 °C. The morphological response to fucoidin is probably not due to its internalization, since the effect is observed at 7 °C where little uptake (3’5 %) occurs. Fucoidin appears to bind at two distinct sites on endothelial monolayers. One site is inhibitable by heparin, while the other site seems to be specific for fucoidin. The observation that fucoidin still induces the retraction of the endothelial cells in the presence of a 100-fold excess of heparin, suggests that binding at the fucoidin-specific site is responsible for the morphological effect of fucoidin. In addition, fucoidin has no detectable effect on monolayers of 3T3 and BHK fibroblast-like cells at 1 mg/ml, 100-fold higher than the concentration required to produce an effect on endothelial cells. Among the possible interpretations of these results is that sulphated fucose-containing glycoconjugates may play a role in the adhesive interactions of endothelial cells.
Spontaneous Turning Behaviour By Dictyostelium Discoideum Slugs
Authors: Fisher P. R. , Grant W. N. , Dohrmann U. , Williams K. L.
Published: April 2021

Dictyostelium discoideum slugs migrating in the absence of external stimuli tend to continue migrating in the original randomly ‘chosen’ direction. Spontaneous deviations from the starting direction accumulate with time at a rate that measures the ‘physiological noise’ in the slugs’ steering mechanism. Most of the spontaneous turning signals arise in the transduction chain ‘downstream’ of the convergence of photosensory and thermosensory processing, and the site(s) of action of the mutation pho-355. A significant amount of steering ‘noise’ arises ‘upstream’ of the site(s) of action of fluoride (which has specific effects on phototaxis and thermotaxis).
Macrocyst Development In Dictyostelium Discoideum : III. Cell-Fusion Inducing Factor Secreted By Giant Cells
Authors: Saga Y. , Yanagisawa K.
Published: April 2021

A factor was discovered that markedly enhances the degree of fusion activity between cells of the opposite mating-type strains, HM1 and NC4, in the sexual cycle of Dictyostelium discoideum. This factor, designated cell-fusion inducing factor (CFIF), was detected initially in supernatants taken from 24 h dark-grown mixed cultures of HM1 and NC4 cells, and subsequently shown to be secreted by giant cells - the fusion products of HM1 and NC4 cells. HM1 cells, cultured in the dark at appropriate temperature, normally acquire fusion-competence specific to NC4 cells. The addition of CFIF to such dark-grown HM1 cultures results in a marked increase in their fusion-competence. In addition, when CFIF is added to light-grown HM1 cultures, in which cells normally do not acquire the ability to fuse with NC4 cells, fusion-competence is induced. The fusion-competence of NC4 cells is unaffected by CFIF, being quite high under the culture conditions used here. Experiments, using actinomycin D, daunomycin and cycloheximide showed that the secretion of CFIF from giant cells requires synthesis of RNA and protein. Possibly, cell fusion triggers production of CFIF, which is rapidly released into the external medium.
Regulation of cytoplasmic streaming in Vallisneria mesophyll cells
Authors: TAKAGI SHINGO , NAGAI REIKO
Published: April 2021

Induction and cessation of the rotational cytoplasmic streaming in Vallisneria mesophyll cells could be controlled by external stimuli. In cells that had been kept in darkness the cytoplasm remained quiescent. However, when the cells were treated in the dark with EGTA solution (10 mM or 20 mM buffered with 10mM-Tris-maleate at pH7·0), rotational cytoplasmic streaming was induced. When the cells were transferred again to artificial pond water in the dark, the induced streaming was inhibited; that is, only 50 % of the observed cells exhibited active streaming after 2 h. When the cells were irradiated continuously with far-red light (λmax = 750 nm, 0·4W/m2) in the same external medium, the induced streaming was inhibited almost completely within 2h.

The relative quantum effectiveness of monochromatic light (450—800 nm) in producing cessation of streaming was also investigated. Irradiation with light of 45 0, 550 and 600 nm was almost as effective as darkness. Light of 500 and 650 nm was less effective than dark exposure. Only irradiation at 750 nm stopped streaming in almost all cells. But when calcium was excluded from the external medium, the effect of far-red light decreased to almost the dark control level. Light of 800 nm also inhibited the streaming but the effect was much less than that of far-red light.

Microfilaments in bundles with the long axis parallel to the streaming direction were localized in the vicinity of the cell membrane. Their configuration, localization and distribution were the same in the present experimental system irrespective of whether the cytoplasm was streaming or quiescent.

Intracellular calcium was examined by electron microscopic cytochemistry and X-ray microanalysis. In cells with streaming induced by EGTA, only a small amount of calcium-containing precipitates formed in the cytoplasm in the presence of antimony. A few precipitates were found in the chloroplasts, the middle lamella of the cell wall and at the border between the cytoplasm and the cell wall. On the other hand, in cells treated with EGTA and subsequently irradiated with far-red light in artificial pond water, many precipitates were observed in the cytoplasm, chloroplasts, mitochondria and endoplasmic reticulum. The middle lamella was also heavily stained.

On the basis of these observations, it was concluded that rotational cytoplasmic streaming in Vallisneria cells can be induced when the free calcium concentration in the cytoplasm decreases and that the induced streaming is arrested when the free calcium concentration in the cytoplasm increases. Far-red light accelerates the increase of calcium in the cytoplasm.
Three-dimensional intranuclear dna organization In situ: three states of condensation and their redistribution as a function of nuclear size near the G1-s border in hela S-3 cells
Authors: Belmont A. , Kendall F. M. , Nicolinif C.
Published: April 2021

Characteristic variations in nuclear morphology occurring with variations in the physiological state of the cell have been observed in a number of systems to date. In this paper, we have critically examined the relationship between nuclear morphology and intranuclear DNA organization near the G1–S transition in HeLa S–3 cells, by the study of both the spatial distribution of optical density values and the optical density histograms for individual Feulgen-stained nuclei. Our results demonstrate that the majority of the DNA is located in a narrow shell surrounding the nuclear and nucleolar borders, and present evidence for at least three discrete states of chromatin condensation. Greater than 90% of the genome appears distributed among the two classes with larger density, and a redistribution between these two classes occurs as a function of changing nuclear size. Numerical simulations indicate that the observed distribution does not arise as an artifact related to overlapping but, in fact, actually represents discrete states of condensation. Interestingly, the extrapolated nuclear area at which the fraction of DNA in the state of highest density is reduced to zero, corresponds closely to the nuclear size shown elsewhere as representing the critical size that HeLa S-3 nuclei must exceed in order to initiate S phase.
Video digitizer analysis of birefringence along the lengths of single chromosomal spindle fibres II. Crane-fly spermatocyte chromosomal spindle fibres are not temperature-labile
Authors: Schaap Catherine J. , Forer Arthur
Published: April 2021

Retardations were measured along the lengths of single chromosomal spindle fibres, from metaphase through anaphase, from video-taped images of crane-fly spermatocytes incubated at various temperatures (4–30°C). These measurements were made using a video digitizer interfaced to a microcomputer. Over most of the range of temperatures at which normal anaphase movement occurs the chromosomal spindle fibres are not temperature-labile. The non-specific and continuous fibre birefringence is temperature-labile, however. The data are discussed with respect to the ‘dynamic equilibrium’ model of anaphase chromosome movement. We conclude that, since single chromosomal fibre birefringence is not temperature-labile over most of the range of temperatures at which normal anaphase chromosome movement occurs, these data do not support the dynamic equilibrium model of anaphase chromosome movement.
Activity and stability of centrosomes in chinese hamster ovary cells in nucleation of microtubules in vitro
Authors: Kuriyama Ryoko
Published: April 2021

Mitotic centrosomes were prepared from Chinese hamster ovary cells and their capacity to nucleate microtubules in vitro was demonstrated by incubation with exogenous brain microtubule protein. The number of microtubules polymerized onto centrosomes was directly counted by electron microscopy of whole-mount preparations. This simple and accurate quantitative assay has permitted characterization of the microtubule nucleating activity of centrosomes in vitro.

The number of microtubules polymerized onto centrosomes varied according to the structure of the centrosome. The activity was roughly proportional to the centriole number. The number and length of microtubules nucleated by centrosomes depended both on the concentration of tubulin and the incubation time with tubulin. Under saturating conditions, an average of 200–2 50 microtubules were initiated by single centrosomes. Centrosomal activity is unstable ( = 8h) and could easily be irreversibly disrupted by a medium of high ionic strength. The activity is stabilized by the addition of glycerol. Centrosomes can be stored at —80°C. The optimum pH for microtubule nucleation is 6·8. Activity is sensitive to protease digestion, but neither DNase or RNase affected the nucleating activity of centrosomes. The activity is temperature-sensitive, but addition of phenylmethylsulphonyl fluoride (PMSF) induces thermal stability. At an optimal concentration of 0·5 mg/ml, this drug increased the half-life of the activity ( = 95 h) and made it resistant to salt extraction. Protease inhibitors other than PMSF or dansyl fluoride did not have any stabilizing effect on the activity.

The difference between the centrosomal structure of polymerized microtubules in vivo and in vitro is discussed.
Deficiency in intercellular communication in two established renal epithelial cell lines (LLC-PK1 and MDCK)
Authors: Sepúlveda F. V. , Pearson J. D.
Published: April 2021

We have studied the cell-to-cell passage of uridine nucleotides in two renal epithelial cell lines (LLC-PK1 and MDCK) and in porcine aortic endothelial cells (PAE). All three cell types incorporated tritiated uridine. After a 3 h incubation the radioactivity was predominantly in the form of acid-soluble compounds, mainly UTP. Prelabelled LLC-PK1 or MDCK cells were unable to transfer radioactivity to added adjacent, non-labelled cells, whereas PAE cells readily formed communicating intercellular junctions, as judged by autoradiographic analysis after a 3h co-culture period. Cell-to-cell communication m either of the renal cell lines was not promoted by treatment with dibutyryl cyclic AMP and methylisobutylxanthine. Radioactivity incorporated into the acidinsoluble pool was not available for intercellular transfer, as assessed in experiments in which cells were prelabelled 24 h before co-culture.
Association Of An Integral Membrane Protein With Glucose Transport And With Anion Transport
Authors: Banyard M. R. C. , White M. K.
Published: April 2021

A monoclonal antibody that recognizes a cell-surface glycoprotein associated with glucose transport was reported previously. Additional information about the function and intracellular distribution of the antigen recognized by this antibody is presented. The monoclonal antibody recognizes a cell-surface and a cytoplasmic determinant. The density of the cell-surface determinant is heterogeneous within the cell population. The subpopulation of cells that carry the cell-surface determinant at high density correspond with a subset of cells that incorporate 2-deoxy-D-[3H]glucose more rapidly than the population as a whole. The monoclonal antibody is used, with cell-affinity chromatography, to isolate this subset of cells.

The cytoplasmic determinant, to which the antibody binds, is associated with the cytoplasmic microfilaments but the antibody is not absorbed by actin. The cell-surface and cytoplasmic components are not identical since the apparent affinity of the antibody for each site is different. The portion of the antigen in the membrane behaves as an integral membrane protein while the remainder is tightly associated with the detergent-insoluble cytoskeleton.

The expression of the antigen on the cell surface is modified by covalent attachment of an inhibitor of anion transport, 4,4’-diisothiocyano-2,2’-disulphonic stilbene. The possible interaction of the anion/lactate transporter with the glucose transporter is discussed.
Kinetic Parameters Of Neutral Amino Acid Transport In Hybrids Between Malignant And Non-Malignant Cells
Authors: White Martyn K.
Published: April 2021

The kinetics of neutral amino acid transport were examined in isogeneic matched pairs of hybrid cells, one member of each pair being tumorigenic, the other not. The L system of transport, which is sodium-independent, was measured by the uptake of leucine, and the A system, which is sodium dependent, by the uptake of methylaminoisobutyric acid. Although there was variation from one cell type to another in both Km and Vmax for the transport of these amino acids, no systematic change was found to be associated with tumorigenicity. This is in marked contrast with hexose uptake where tumorigenicity was invariably found to be associated with a reduction in Km. It thus appears that whatever molecular change is responsible for the alteration in the kinetics of hexose transport, it is specific, at least to the extent that it does not affect either sodium-dependent or sodium-independent transport of neutral amino acids.