bluuebunny/crossref_metadata_2025 huggingface dataset:
Proteins of the membrane skeleton in rat sertoli cells
Authors: Ziparo E. , Zani B. M. , Filippini A. , Stefanini M. , Marchesi V. T.
Published: April 2021

Analogues of the α, β and γ subunits of human spectrin and erythroid protein 4.1 have been detected in rat Sertoli cell primary cultures. Immunofluorescence of permeabilized cells showed that erythroid type spectrin, protein 4.1 and actin co-distribute within the cells as filamentous structures. Fodrin-like molecules were distributed in a diffuse manner, mostly associated with the plasma membrane. Immunoprecipitation and immunoblotting experiments indicated that the polypeptides present in rat Sertoli cells are immunologically related and display molecular weights similar to their analogues in the human erythroid and non-erythroid membrane skeleton.
Electron-microscopic studies of the ca antigen, epitectin
Authors: Bramwell M. E. , Wiseman G. , Shotton D. M.
Published: April 2021

Epitectin, the mucin-like glycoprotein defined by the monoclonal antibodies CAI, CA2 and CA3, has been examined by electron microscopy to determine its shape and size. It appears to be a single extended strand with a mean length of about 270 nm. The antibodies CAI and CA2 appear to bind preferentially to a terminal site on the epitectin molecule.
Cytochemical and biochemical evidence of cathepsin B in malignant, transformed and normal breast epithelial cells
Authors: Křepela E. , Bártek J. , Skalková D. , Vičar J. , Rasnick D. , Taylor-Papadimitriou J. , Hallowes R. C.
Published: April 2021

Human breast cancer cell lines, as well as transformed mammary epithelial cells (HBL-100) and growth-stimulated normal breast epithelial cells showed positive cytochemical reaction with the proteinase substrate 2-(N-benzyloxycarbonyl-L-arginyl -L -arginylamido) - 4 -methoxynaphthalene, in the presence of 5-nitrosalicylaldehyde. The reaction product, small fluorescent granules, was distributed throughout the cytoplasm, in the perinuclear zone, in some cytoplasmic projections, and at the cell surface. Using a panel of various proteinase inhibitors, we found that the formation of the reaction product was an enzymic function of a cysteine proteinase.

Using the substrate 7-(N-benzyloxycarbonyl-L-arginyl-L-arginylamido)-4-methylcoumarin, we evaluated some biochemical properties of the cysteine proteinase, including pH-activity profile, pH stability, apparent relative molecular mass and sensitivity toward various proteinase inhibitors. We found that the proteinase from the studied breast epithelial cells exhibited characteristics of a mature form of cathepsin B.

Taken together, the cytochemical and biochemical data provide evidence that human breast epithelial cells of cancer origin, as well as in the transformed or growth-stimulated state express active cathepsin B and compartmentalize it into specific subcellular sites.
Chemotaxis towards pteridines during development of Dictyostelium
Authors: Tillinghast H. S. , Newell P. C.
Published: April 2021

Following a previous study indicating a sensitivity to folate during the developmental phase of Dictyostelium discoideum, a series of pteridines were investigated for their ability to induce amoebal chemotaxis during development of this organism. Several compounds were found to resemble folate in their ability to induce chemotaxis of both vegetative amoebae and amoebae developing during the first few hours of starvation. One compound, L-monapterin, was distinct in showing chemotactic activity only during the developmental phase after approximately 12 h of starvation. Tests using the polymerization of cytoskeletal actin as an assay for a cellular response correlated with chemotaxis showed that 10 nM-L-monapterin was a potent inducer of this response and that responsiveness appeared only after 12 h of development. The timing of these events may be correlated with the formation of tips containing stalk cells that occurs in multicellular aggregates at approximately 12 h, and suggests a role for L-monapterin (or a naturally occurring, closely related pteridine) in cell sorting. The evolutionary significance of the roles of pteridines is discussed.
Tissue inhibitor of metalloproteinases (timp) regulates extracellular type i collagen degradation by chondrocytes and endothelial cells
Authors: Gavrilovic Jelena , Hembry Rosalind M. , Reynolds John J. , Murphy Gillian
Published: April 2021

A specific antiserum to purified rabbit tissue inhibitor of metalloproteinases (TIMP) was raised in sheep, characterized and used to investigate the role of TIMP in a model system. Chondrocytes and endothelial cells cultured on 14C-labelled type I collagen films and stimulated to produce collagenase were unable to degrade the films unless the anti-TIMP antibody was added. The degradation induced was inhibited by a specific anti-rabbit collagenase antibody. It was concluded that TIMP is a major regulatory factor in cell-mediated collagen degradation.
Gland formation of human colon cancer cells combined with foetal rat mesenchyme in organ culture: an ultrastructural study
Authors: Fukamachi Hiroshi , Mizuno Takeo , Kim Young S.
Published: April 2021

The morphogenetic potential of human colon cancer cells was examined by combining cells with foetal rat mesenchyme in organ culture. Out of four cell lines examined, LS174T cells formed glandular structures composed of a simple columnar or cuboidal epithelium with a lumen in the centre of the cell mass. Ultrastructurally, these gland-forming LS174T cells exhibited polarity, with nuclei located basally, microvilli projecting into the lumen, tight junctions at the cellcell junction facing the lumen, desmosomes at the subluminal region, and basal laminae at the epithelial-mesenchymal interface. Other cell lines did not form such glandular structures, while some cells could form microvilli, tight junctions or basal laminae, which were arranged randomly. It is concluded that it is not the existence but the ordered arrangement of these structures that is essential for gland formation by epithelial cells. The mechanism of gland formation is discussed with special reference to the arrangement of different structures.
Expression of C-myc during differentiation of the human teratocarcinoma cell line Tera-2
Authors: Schofield P. N. , Engstrom W. , Lee A. J. , Biddle C. , Graham C. F.
Published: April 2021

The quantity of c-myc mRNA was measured during the retinoic-acid-induced differentiation of the pluripotent human teratoma cell line, Tera-2 cl. 13. As the cells were exposed to retinoic acid for longer periods of time the duration of the cell cycle progressively increased (measured by the rate of S phase entry) until the cells were effectively quiescent and expressed characteristic differentiation markers. Under these circumstances steady-state levels of c-myc expression increased by up to 1·6-fold with respect to rapidly growing undifferentiated cells. Southern blot analysis of the c-myc genes in Tera-2 indicated no major rearrangement or amplification in the cell line.
Leukotrienes stimulate initiation of DNA synthesis in cultured arterial smooth muscle cells
Authors: Palmberg Lena , Claesson Hans-Erik , Thyberg Johan
Published: April 2021

The effects of leukotrienes on initiation of DNA synthesis in growth-arrested arterial smooth muscle cells cultivated in a defined, serum-free medium were studied. The results showed that LTB4, LTC4 and LTD4 were all stimulatory with a distinct effect already at 0·01 pM and a maximal effect at 10pM; in contrast, LTE4 lacked effect. 5S,12S-DHETE, an isomer of LTB4, was inactive in itself but blocked the effect of LTB4. Treatment of the cells with indomethacin or acetylsalicylic acid, two cyclooxygenase inhibitors, blocked induction of DNA synthesis by LTB4, indicating that the effect of this compound was mediated by a cyclooxygenase product. Up to 10 pM, the leukotrienes stimulated initiation of DNA synthesis with similar potency to platelet-derived growth factor (PDGF). The maximum labelling index obtained with PDGF was, however, about twice that obtained with the leukotrienes. At suboptimal concentrations of PDGF, the leukotrienes had an additive effect. The prereplicative lag phase was 16–20h with LTB4, 12–16 h with LTC4, and 8—12 h with PDGF. In metabolic experiments no signs of synthesis of leukotrienes were detected by cells stimulated with the calcium ionophore A23187, arachidonic acid, or LTA4 for 10–30 min, neither was any apparent degradation of LTB4 observed. On the other hand, LTC4 was transformed into LTD4 and LTE4. Taken together, the results indicate that leukotrienes are able to stimulate quiescent arterial smooth muscle cells to enter the cell cycle and to replicate their DNA. In vivo, stimulation of cell growth by leukotrienes could add to other established functions of these substances in tissue repair, inflammation, and atherogenesis.
Transfection and stable expression of a dominant selective marker Ecogpt in a cultured cell line of the fish, Carassius auratus
Authors: Isa K. , Shima A.
Published: April 2021

The synthetic plasmid, pSV2-gpt, was transfected into the cultured fish cells (RBCF-1 line) using polycation and dimethyl sulphoxide. The maximum transfection frequency estimated by colony number in the selection medium was 585 trans-fectants/5× 10s treated cells per 50 ng plasmid DNA. The isolated transfectants expressed the xanthine guanine phosphoribosyltransferase (XGPRT) activity encoded by the plasmid DNA associated with the promoter of simian virus 40 (SV40). The resistance to mycophenolic acid and the XGPRT activity of every transfectant examined were stable, indicating the possibility that pSV2-gpt was integrated into the genomic DNA of the host fish cells. Our results, in addition to those already reported for mammalian and avian cell lines, indicate that the promoter in the early region of SV40 can function in cultured fish cells. This success in obtaining cultured fish cells with a dominant selective marker will provide a useful clue for somatic cell genetic studies of fish in the future.
Gradient perception by neutrophil leucocytes, continued
Authors: Lauffenburger Douglas , Farrell Brian , Tranquillo Robert , Kistler Amy , Zigmond Sally
Published: April 2021

The mechanism by which neutrophil leucocytes exhibit chemotaxis continues to be the subject of debate. In recent articles in this journal (Vicker el al. 1986; Haston & Wilkinson, 1987) the question of whether neutrophils can perceive spatial attractant concentration gradients has been discussed. The purpose of this brief note is to give attention to some information that should help clarify some of the points raised by these articles.
Differential expression of the ed sequence-containing form of cellular fibronectin in embryonic and adult human tissues
Authors: Vartio Tapio , Laitinen Lusa , Närvänen Outi , Cutolo Maurizio , Thornell Lars-Erik , Zardi Luciano , Virtanen Ismo
Published: April 2021

Monoclonal mouse hybridoma antibodies were obtained for secreted cellular fibronectin (cFn) from A8387 fibrosarcoma cells. One of them, 52-DH1 (DH), reacted exclusively with cFns but not with plasma Fns (pFns) in immunoblotting and solid-phase EIA. The DH antibody also recognized thermolysin cFn fragments and β-galacto-sidase-Fn fusion protein which contained the ED sequence specific to at least some forms of cFns. On the other hand, the DH antibody failed to recognize a fusion protein that was otherwise identical but lacked the ED sequence. Thus, the antigenic determinant for the DH antibody was located to the ED sequence. The DH antibody was then used to study the expression of ED sequence containing cFn (EcFn). For comparisons, another monoclonal antibody, 52BF12 (BF), recognizing equally well both pFns and cFns, was used. Immunoblotting of pFn fragments indicated that this antibody had the antigenic determinant at or close to the cell-binding site of Fn. EcFn wasrevealed by the DH antibody in embryonic and adult fibroblasts and in a variety of other cultured normal and malignant human cells. In embryonic tissues EcFn was abundant in developing basement membranes, as shown in foetal kidney and muscle, while in adult tissues it was confined only to endothelia of larger blood vessels. Furthermore, in embryonic tissues the capillaries showed bright EcFn-positivity not found any more in adult tissues. Human plasma contained a small quantity of EcFn, which may hence have an endothelial origin. EcFn was also prominent in the stroma of malignant tumours as well as in reactive benign conditions, such as granulation tissue and decidual cells. The results suggest that EcFn is a form of the protein which may have a particular role in developing and reactive tissues in embryos and adults.
F-actin distribution in polymorphonuclear leucocytes
Authors: Haston Wendy S.
Published: April 2021

The polarization and amoeboid locomotion of neutrophil leucocytes is stimulated by chemotactic factors, which initiate waves of contraction in both adherent and non-adherent neutrophils. These cyclical contractile events have previously been analysed by time-lapse filming but the mechanisms involved in the coordination of the cytoskeleton during locomotion have not been elucidated, one reason being because of the problems involved in fixing motile cells. In this paper we show that improved fixation of motile neutrophils with low concentrations of glutaraldehyde followed by glycine quenching demonstrated significant differences in the pattern of staining with TRITC-phalloidin in neutrophils moving on different substrata. Previous film analysis had shown the basic features of locomotion to be similar on all substrata.

A prominent feature of leucocyte locomotion on two-dimensional substrata (e.g. protein-coated glass), on three-dimensional collagen gels or inmotile cells floating in suspension, is the wave of contraction that passes antero-posteriorly along the length of the cell. The organization of the cytoskeletal elements has not been demonstrated at contraction waves, but light fixation with glutaraldehyde followed by staining with TRITC-phalloidin demonstrated prominent bands of Factin in neutrophils inside collagen gels. These bands were not present in neutrophils either in suspension or moving on a two-dimensional substratum.

Although all motile neutrophils had brightly stained anterior lamellipodia, the cells moving on the two-dimensional substratum had very extensively ruffled leading lamellae stained very brightly with TRITC-phalloidin.

The reasons for the absence of consistent bands of F-actin at contraction waves are discussed.
Contact behaviour during the reassociation of dissociated epithelial cells in primary culture
Authors: Brown R. M. , Middleton C. A.
Published: April 2021

The behaviour in culture of dissociated epithelial cells from chick embryo pigmented retina epithelium (PRE), corneal epithelium (CE) and epidermis has been studied using time-lapse cinematography. The analysis concentrated on the contact behaviour of 60 previously isolated cells of each type during a 24h period starting 3–5 h after the cells were plated out.

During the period analysed the number of isolated cells in cultures of all three types gradually decreased as they became incorporated into islands and sheets of cells. However, there were significant differences in behaviour between the cell types during the establishment of these sheets and islands. In PRE cell cultures, islands of cells developed because, throughout the period of analysis, collisions involving previously isolated cells almost invariably resulted in the development of a stable contact. Once having established contact with another cell these cells rarely broke away again to become reisolated. In contrast the contacts formed between colliding CE and epidermal cells were, at least initially, much less stable and cells of both these types were frequently seen to break away and become reisolated after colliding with other cells. Sheets and islands of cells eventually developed in these cultures because the frequency with which isolated cells become reisolated decreased with increasing time in culture.

The possible reasons underlying the different behaviour of PRE cells, when compared with that of CE and epidermal cells, are discussed. It is suggested that the decreasing tendency of isolated CE and epidermal cells to become reisolated may be related to the formation of desmosomes.
The cell cycle and its relationship to development in Acanthamoeba Castellanii
Authors: Stöhr Michael , Bommert Kurt , Schulze Ingrid , Jantzen Helga
Published: April 2021

The cell cycle and the relationship between particular cell cycle phases and the differentiation of trophozoites into cysts were reinvestigated in Acanthamoeba castellanii using flow fluorometric measurements of nuclear DNA content and synthesis, and synchronization of cells by release from the stationary phase. The investigation was performed with cultures growing in non-defined medium (ND cells) showing a high degree of encystation in response to starvation, and with subcultures growing in chemically defined nutrient medium (D cells) exhibiting a very low encystation competence. In both cultures the cell cycle starts with a short S phase taking place simultaneously with cytokinesis, followed by a long G2 phase. A G1 phase seems to be either absent or very short. Synchronization experiments reveal that in ND cells encystation is initiated from a particular position of late G2. The high encystation competence of stationary phase ND cells seems to be due to arrest of cells at this particular cell cycle position. The lack of encystation competence of stationary phase D cells correlates with the loss of accumulation of cells at this particular stage of the cell cycle. This change of the property of cells is related to the growth condition and not to an irreversible loss of encystation competence of D cells.
Taxol-induced structures derived from cytoskeletal elements of the Nicotiana pollen tube
Authors: Tiezzi A. , Moscatelli A. , Milanesi C. , Ciampolini F. , Cresti M.
Published: April 2021

Nicotians pollen tube extracts were treated with 20 μM-taxol and structures derived from the cytoskeleton were collected and observed in the electron microscope. We have identified three different groups of elements with characteristic size and morphology: wide filaments (WF) probably formed by assembled microtubules, thin filaments (TF) interacting with WF and debrislike material principally associated with TF. SDS-PAGE and Western blot analysis provide some preliminary evidence of the biochemical composition of the taxol-induced structures.
Light-induced colour changes by the iridophores of the neon tetra, Paracheirodon Innesi
Authors: Clothier Jennifer , Lythgoe J. N.
Published: April 2021

The iridophores of the Neon tetra Paracheirodon innesi consist of alternating layers of guanine and cytoplasm. In the dark-adapted state the reflected light from constructive interference is in the ultraviolet or blue. When exposed to light the cytoplasm layers increase in thickness and as a result the reflections shift to longer wavelengths and the iridophores appear green. The iridophores are thought to contain a rhodopsin-like molecule and we suggest that the colour-change mechanism involves the light-induced opening of sodium channels in the plasma membrane, leading osmotically to an increase in thickness of the cytoplasm layers. Experimental support for this suggestion was obtained by the substitution of choline chloride for sodium chloride in the perfusing medium, which can be done without altering the osmotic strength of the perfusing medium. This procedure almost abolished the light response and makes it seem likely that sodium ions are necessary for the light response to take place.
Induction of cybrid strains of Tetrahymena thermophila by electrofusion
Authors: Gaertig Jacek , Kiersnowska Mauryla , Iftode Francine
Published: April 2021

In this paper, the electrofusion of the ciliated protozoan, Tetrahymena thermophila, is described. Deciliated cells were brought into close membrane contact by dielectrophoresis in a weakly conductive medium. Then cell fusion was induced by application of repeated electric pulses. Up to 20 prestarved, logarithmic or stationary phase cells of the same or different mating types may form a single giant cell. The polykaryons are fully able to regenerate cilia and became motile. After an initial process of partial integration the polykaryons yield viable clones by separation of components. Cytoplasmic exchange between fused components occurs before separation. Cytoplasmically inherited chloramphenicol resistance was transmitted from one strain to another by electrofusion and drug-resistant cybrid strains were generated.
Cloning of a gene encoding an antigen associated with the centrosome in Drosophila
Authors: Whitfield W. G. F. , Millar S. E. , Saumweber H. , Frasch M. , Glover D. M.
Published: April 2021

The monoclonal antibody Bx63 recognizes a centrosomal antigen of Drosophila melanogaster by indirect immunofluorescence and identifies two proteins, with apparent molecular weights of 185 ×103 and 66×103, on Western blots. We have used this antibody to isolate five clones (λcsl, -2, -3, -4 and λj63) from λgtll expression libraries of Drosophila DNA. Using polyclonal anti-centro- somal sera raised against both immunoaffinity- purified Bx63 antigen and electrophoretically purified fusion protein from clone λcs3, we have demonstrated that the fusion proteins encoded by four of these clones (λcsl-4) share at least two epitopes with the 185×103Mr centrosomal antigen. This indicates that clones λcsl-4 contain DNA from the gene coding for this protein. The four clones are independent isolates from a single chromosomal site, which we show by in situ hybridization to correspond with salivary gland chromosome region 88E 4—8. A low-abundance transcript of approximately 4·0×103 bases corresponding to the cloned gene is detected in all stages of the Drosophila life-cycle.
The mechanism of secretion of the milk fat globule
Authors: Wooding F. B. P.
Published: April 2021

The morphology of secretion of the fat globule is identical in goat, guinea-pig and cow. The smallest fat droplets, which are found in the basal cytoplasm of the secretory cell, have no membrane separating the lipid from the cytoplasm and no direct association with rough endoplasmic reticulum. In the apex of the cell, fat droplets have numerous peripheral vesicles, most of which appear to be derived from the Golgi body. The progressive fusion of these vesicles results in the extrusion of the fat droplet surrounded by a unit membrane originating partly from the originally peripheral vesicles and partly from the plasmalemma. This membrane bears on its inner surface a zone of dense material which appears to be derived from the cytoplasm, and this is also seen around fat globules in secreted milk. Thus the term apocrine secretion is considered a valid description of the process.
Two populations of mast cells on fibroblast monolayers: Correlation of quantitative microscopy and functional activity
Authors: Hammel I. , Shiloh-Rabinovich H. , Nir I.
Published: April 2021

We have previously shown that a confluent layer of mature mast cells is obtained when lymph node cells are grown on embryonic flbroblast monolayers. Two populations of mast cells may be observed, depending on treatment of the mice from which the lymph node cells are derived. We report now on the morphometric evaluation of these two mast cell entities, and we correlate this with cellular biochemistry and secretory behaviour. The first type of mast cell is small (265 ± 20 μm3). It arises from the embryonic monolayer and the cells have feathers resembling those of connective tissue mast cells. These cultured cells are filled with about 1000 homogeneous electron-dense granules, which usually range in diameter from 0·05 to 0·2 μm. The second type of mast cell arises from precursors originating from the lymph node and they have feathers of mucosal mast cells. These cells are larger (480 ± 40 μm3) and contain about 300 heterogeneous granules, which range from 0·1 to 0·8; μm. Both cell entities contain about equal amounts of histamine, serotonin and chymase. Biologically, the two cell entities secrete soluble mediators (histamine and serotonin) at different rates compared to the rate at which they secrete chymase. We suggest that such a pattern of secretion exhibits a form of degranulation that permits the release of freely diffusible mediators that are loosely bound to granules, but only partially permits the secretion of insoluble mediators, which are stored in the granules. Alternatively, there might be a mechanism that rapidly inactivates or binds chymase so that only the vasoamines will be free.
The formation of siliceous scales by Raphidiophrys ambigua (protista, centroheliozoa)
Authors: Patterson David J. , Dürrschmidt Monika
Published: April 2021

Ultrastructural aspects of the formation of siliceous scales by the protozoon Raphidiophrys ambigua are described. Cells in culture become scale-free in silicon-impoverished medium. Scales may be seen adhering to the cell surface within four to six hours of the addition of silicon to the medium. Silica scale formation occurs within deposition vesicles (SDVs) near the periphery of the cell. Many scales are formed at the same time; each may be in a different phase of scale formation. The source of the SDV membrane is not known. Silicification proceeds centrifugally from two pattern centres. The first parts of the forming scale are two opposed sterna,which establish the longitudinal axis of the scale. One sternum develops from each pattern centre. The scale develops outwards by formation of fairly evenly spaced lateral ribs from each central sternum. Distally, the ribs branch to form a reticulate pattern. A thin continuous sheet of silicon is added to the periphery of the scale, curving inwards to form a rim. The development of this pattern may be described in terms of a small number of morphogenetic processes.
High frequencies of preprophase bands in soybean protoplast cultures
Authors: Wang Hong , Cutler Adrian J. , Fowke Larry C.
Published: April 2021

Protoplasts derived from a non-regenerable soybean cell line provide an excellent experimental system for studying plant preprophase bands (PPBs). Cultured protoplasts developed PPBs in high frequencies, permitting a detailed analysis of PPB development. From observations of thousands of PPBs, six distinct developmental stages were identified. This classification should prove useful in recognizing developmental stages of PPBs and in comparing results among different tissues and species. Perinuclear fluorescence appeared when PPBs were well developed. It consisted of microtubule strands radiating from the nuclear surface at its early stages and more extensive arrays on the surface at later stages.

All protoplast cultures from O·5 day to 5 days had a PPB index (ratio between number of PPBs and number of phragmoplasts) greater than unity, suggesting that in this cell line a high frequency of PPBs is apparently not related to the potential for organized growth (e.g. embryogenesis). Different methods of quantifying the occurrence of PPBs were evaluated. The PPB value (ratio between % of cells with PPB and the mitotic index) was compared with the PPB index and results indicate that it is a useful new parameter for studies of the distribution of PPBs in plant cells.
Ductal epithelial cells cultured from human foetal epididymis and vas deferens: relevance to sterility in cystic fibrosis
Authors: Harris Ann , Coleman Lindsay
Published: April 2021

A tissue culture System for epithelial cells derived from male human foetal genital ducts has been established. The cells show morphological and biochemical characteristics of ductal epithelial cells, and can be passaged and maintained in culture for considerable periods of time. These cells will provide a suitable System for investigating, by electrophysiological, biochemical and molecular biological methods, the cause of sterility in cystic fibrosis.
Red cell membrane protein distribution during malarial invasion
Authors: Dluzewski A. R. , Fryer P. R. , Griffiths S. , Wilson R. J. M. , Gratzer W. B.
Published: April 2021

Immuno-gold labelling electron microscopy of thin sections was used to determine the distribution of red cell membrane and membrane skeleton proteins in the vicinity of internalized malaria parasites. When examined immediately after invasion (young ring-stage parasites), the parasitophorous vacuole membranes of both Plasmodium falciparum and P. knowlesi were found to be characterized by the essentially complete absence of spectrin, ankyrin and the most abundant transmembrane protein, band 3. P. knowlesi merozoites were trapped in the attached but not internalized state by pretreatment with cytochalasin B. In this merozoitered cell complex antibody labelling showed that band 3 had been eliminated from the region of the host cell membrane in contact with the parasite. Internal vesicles, originating apparently from the site of attachment, were often observed in the red cell. Opposite the attached parasite a cavity was also sometimes seen in the host cell, presumably representing an incipient internal vesicle. The membrane was intact, as judged by the absence of protein (haemoglobin) in the cavity, and, like the membranes surrounding the internal vesicles, was devoid of membrane proteins. A large multilamellar body was sometimes seen in the merozoite close to its point of attachment. The lamellar spacing was about 50 nm. The electron microscope images suggest a diffusion of electron-dense material from the lamellar body into the cavity in the host cell.
Internalization and recycling to serotonin-containing granules of the 80K intégral membrane protein exposed on the surface of secreting rat basophilie leukaemia cells
Authors: Bonifacino Juan S. , Yuan Lydia , Sandoval Ignacio V.
Published: April 2021

The 80K (80x103Mr) integra! membrane protein, first described in the secretory granules of rat basophilie leukaemia (RBL) cells, is also localized to lysosomes in these cells. The protein displays the same distribution in natural killer lymphocytes (RNK-7), wherein it codistributes with cytolysin in secretory granules. In contrast, the protein is absent from the endocrine and exocrine secretory granules of rat pancreatic acinar and pituitary cells, respectively, where it is confined to lysosomes. The protein colocalizes with lysosomal intégral membrane proteins in ail the cells studied, indicating that is largely restricted to secretory granules with lysosomal properties (LSG) and lysosomes. The protein expressed on the surface of secreting RBL cells is internalized by endocytosis via coated pits, and found in coated vesicles, endosomes, multivesicular bodies and Golgi System, before being recycled to LSG and partly delivered to lysosomes. The recycled protein is re-expressed on the surface of cells stimulated to secrete a second time.
Brain dynein crossbridges microtubules into bundles
Authors: Amos Linda A.
Published: April 2021

Cytoplasmic dynein was purified from pig brain, using a modified version of published procedures, in order to study its interaction with microtubules. Since the preparation produces ATP-dependent sliding of taxol-stabilized purified microtubules over glass and runs on SDS-containing gels as a major band exceeding 300 000 Mr plus a medium chain band at about 75 000Mr, it is assumed to be identical to the mammalian brain dynein (MAP 1C) purified by Vallee and colleagues.

When viewed by electron microscopy in negative stain, individual particles show two distinct configurations. Some are clearly similar to the twoheaded bouquet structure already shown for MAP 1C. A larger number of molecules in the present preparation appear to have the two heads fused together, forming a dimeric globular particle with two separate tails. They are referred to as phi-particles, because of their resemblance to the greek letterϕ.

A model for the structural relationship between the two molecular forms is presented. The stems of two associated dynein subunits may separate beyond the base, to form a bouquet, or they may remain fused to form the larger tail of a phi-particle. The smaller tail probably represents a combined pair of features equivalent to the ‘stalks’ shown to emanate from axonemal dynein heads by Goodenough and colleagues. Both tails of a phi-particle can bind to microtubules, even in the presence of ATP, and cause microtubule bundling. These results suggest a complete structural homology between axonemal and cytoplasmic dynein.
The organization of tip-growth-related organelles and microtubules revealed by quantitative analysis of freeze-substituted oomycete hyphae
Authors: Heath I. Brent , Kaminskyj Susan G. W.
Published: April 2021

The distribution of organelles and microtubules in hyphal tips of the oomycete, Saprolegnia feras, were quantitatively determined at high resolution from serial-section electron microscopy of freeze-substituted cells. All the organelles and the microtubules were non-uniformly distributed, each showing a characteristic longitudinal gradient starting at a different point behind the tip. In addition, when the cytoplasmic cross-sectional area was divided into radial regions, all organelles occurred preferentially in either the central (mitochondria and Golgi bodies) or the peripheral (microtubules, wall vesicles and spherical vesicles) region. The nuclei were so large as to span both regions but were always oriented with their centrioles facing the plasmalemma. Microtubules occurred in the extreme tips, became more abundant sub-apically, were predominantly short but increased in mean length with distance from the tip. The correlated patterns of organelle and cytoskeleton organization from this and previous work show that neither the microtubules nor the detected arrays of actin are sufficient to account for most organelle arrangements. However, on the basis of the distribution and orientation of the predominantly elongated wall vesicles, we suggest that the wall vesicles travel radially from their origin at the centrally located Golgi bodies to the cell periphery where they are transported longitudinally to the hyphal tip in conjunction with the plasmalemma-associated actin cables. Our data also suggest that the hyphae contain a cortical ectoplasm with which the nuclei interact, at least in part, via their centrioles and centriole-associated microtubules, and whose mechanical integrity is increased by both the peripheral actin cables and a high density of microtubules. We suggest that the endoplasm is less strong and has physiological properties that enhance the differentiation of endoplasmic reticulum and nuclear envelope into Golgi body production.
A Ca2+-sensitive glycoprotein, GP90, associated with the cytoskeleton from brain and gizzard
Authors: Moss Diana J. , White Carol A.
Published: April 2021

We describe here the expression during development, tissue distribution and molecular properties of GP90: a major concanavalin A (ConA)-binding glycoprotein present in the neuronal membrane skeleton from chicken brain. GP90 is co-isolated with, and has a similar developmental profile to contactin (previously called GP130). In whole brain, GP90 undergoes rapid synthesis between embryonic days 10 and 12. Unlike contactin, it is not restricted to nervous tissue and is quite abundant in gizzard, where there are two antigenically related proteins of 100K and 90K (K = 103Mr). In both brain and gizzard GP90 and (GP100) are enriched in the membrane skeleton fraction. Trypsinization of live cells suggests that GP90 from gizzard is related to GP100 by the removal of a polypeptide chain. GP90 from both neurones and gizzard cells is protected from proteolysis by the presence of extracellular Ca2+. In the absence of Ca2+ a soluble fragment of approximately 70K can be released from the surface of cells indicating that a large fraction of GP90 is extracellular. Deglycosylation of GP90 from brain using endoglycosidase F demonstrates the presence of at least five carbohydrate chains and a polypeptide chain of approximately 80K. Immunofluorescence studies show that GP90 is exposed on the surface of cultured neurones, gizzard cells and most glial cells with the exception of Schwann cells. It is observed in clusters or patches even when cells are prefixed, suggesting this may be the normal distribution of GP90.
Genetic Elements in Escherichia coli
Authors: Sharpe Colin R.
Published: April 2021

This book is directed toward the teaching of advanced undergraduate courses and covers the genetics and, in part, the molecular biology of plasmids and phage in Escherichia coli. It will appeal, however, to a wider audience, that is to anyone whose research concerns plasmids and phage either directly or superficially as ‘reagents’ in molecular biology. Within the scope of this book lie such topics as gene mapping, recombination, DNA repair and transposition. The section on phage lambda provides a good if somewhat limited introduction to this important system, which is a fault that may be excused given the excellent concise nature of the book, which conforms to the aim of the Series to provide ‘authoritative texts of a manageable size’.
Fibroblast contractility and actin organization are stimulated by microtubule inhibitors
Authors: Danowski Barbara A.
Published: April 2021

Despite considerable evidence that cytoplasmic microtubules play some role in guiding or controlling the locomotion of tissue cells, the nature of this control is not understood. In particular, little is known about the role of microtubules in the exertion of propulsive ‘traction’ forces, or about microtubule effects on the organization of the cytoplasmic actin stress fibers. In this study, the silicone rubber substratum technique was used in combination with fluorescence microscopy in order to observe the effects of microtubule-depolymerizing drugs on the contractile strength and organization of cytoplasmic actin networks.

Perfusion with a variety of microtubule poisons (either colcemid, nocodazole or vinblastine) was found to cause a rapid and substantial strengthening of fibroblast contractility. This was demonstrated in two established fibroblast cell lines, as well as in primary cultures of rat gingival fibroblasts and embryonic chick heart fibroblasts. Treatment with the drug taxol, which promotes microtubule assembly, was found to prevent the strengthening effects of the microtubule inhibitors. It was also found that the disruption of actin stress fibers by the phorbol ester tumor promoter, TPA, is reversed by microtubule poisons: stress fibers reform within 30 min of the addition of the microtubule drugs, despite the continued presence and activity of the TPA.

Several possible mechanisms are considered, including the idea that microtubule assembly normally exerts a pushing force, which counterbalances part of the contractile force exerted by the actin stress fibers. However, the mechanism that seems best to account for the observations is that microtubules modulate, in an inhibitory fashion, the contractility and the state of organization of cytoplasmic actin.
Sertoli cell ectoplasmic specializations: a type of actin-associated adhesion junction?
Authors: Grove B. D. , Vogl A. W.
Published: April 2021

In this paper we provide evidence that ectoplasmic specializations are a form of intercellular adhesion junction. Ectoplasmic specializations, found at basal junctions between adjacent Sertoli cells and at sites of adhesion between Sertoli cells and germ cells, consist of actin filament bundles sandwiched between the plasma membrane and a cistern of endoplasmic reticulum. The actin filaments in each bundle are unipolar and are hexagonally packed. The bundles are coupled to the adjacent membranes and to each other. Because ectoplasmic specializations are associated with junctional sites, they may play a role in intercellular adhesion. In this study, we report a procedure for obtaining samples enriched for ectoplasmic specializations and identify polypeptides that may be associated with ectoplasmic specializations. On SDS-poly-acrylamide gels, an 83K (K = 103 Mr) polypeptide is specific to the ectoplasmic specialization-enriched sample, sμggesting that it may be a component of ectoplasmic specializations. Other polypeptides at 38, 53, 56 and 69K also may be associated with ectoplasmic specializations. Immunoblots further indicate that fimbrin and vinculin are present in the ectoplasmic specialization-enriched fraction. In addition, immunofluorescence indicates that vinculin is associated with spermatid-Sertoli cell and Sertoli-Sertoli cell junctions. We suspect that fimbrin, an actin-bundling protein, may be involved in cross-linking the hexagonally packed actin filaments in ectoplasmic specializations while vinculin may be associated with actin—membrane linkages. If so, ectoplasmic specializations may be a new class of actin-associated junctional site. Moreover, the presence of vinculin in testicular fractions enriched for ectoplasmic specializations and at junctional sites supports the view that these structures may play a role in intercellular adhesion, possibly by stabilizing an adhesive membrane domain.
Evidence for a second class of membrane glycoprotein involved in cell adhesion
Authors: Norris Wendie E.
Published: April 2021

It is believed that transmembrane relationships exist between the cytoskeleton and the extracellular matrix through integral membrane proteins, almost certainly glycoproteins, which would act as transmembrane receptors. Such receptors would include those involved in cell adhesion. I have been able to isolate a detergent-soluble fraction from chick embryo fibroblasts that is enriched in these integral membrane proteins by making use of their amphipathic character to phase-separate them in the detergent Triton X-114. Antisera raised to this fraction had biological activities interfering with cell adhesion and motility. A 45×103Mr glycoprotein unique to this fraction appears to be responsible for this biological activity and is a candidate for a transmembrane receptor involved in cell adhesion.
Three-dimensional analysis of morphogenesis induced by mating pheromone α factor in saccharomyces cerevisiae
Authors: Baba Misuzu , Baba Norio , Ohsumi Yoshinori , Kanaya Koichi , Osumi Masako
Published: April 2021

Ultrastructural analyses of cytoplasmic changes in Saccharomyces cerevisiae X2180-1A (MATa) that had been treated with α factor were performed by using the freeze-substitution fixation method. After α factor treatment, cells exhibited a pointed projection, which is a unique pattern of oriented cell surface growth. The relationship between projection formation and intracellular organelles was examined using serial thin sections and computer-aided three-dimensional reconstructions. Using these analyses membrane vesicles and other organ-elles were detected, and studies on their dynamic structural reorganization became feasible. Production of membrane vesicles (average 65 nm in diameter) was induced upon exposure of the cells to afactor before projection emergence. The total number of membrane vesicles increased at the early stage and decreased at the late stage of projection formation. Three-dimensional analysis indicated that the vesicles were at first dispersed throughout the cell, then accumulated at the site where the projection formed. Morphological changes and multiplication of the Golgi body were seen during the process of projection formation. Other intracellular organelles (nucleus, vacuole, rough endoplasmic reticulum and mitochondria) were also rearranged, showing a polar organization of the cytoplasm during projection formation.
Mechanically cut mitotic spindles: clean cuts and stable microtubules
Authors: Nicklas R. Bruce , Lee Greta M. , Rieder Conly L. , Rupp Gerald
Published: April 2021

We have discovered an easy way to cut through the mitotic spindle at any desired place. Spindles of demembranated cricket or grasshopper spermatocytes were severed with a microneedle between the chromosomes and one pole, and the cut-off polar piece was swept away. Spindle structure and microtubule dynamics in cut spindles were studied by anti-tubulin immunostaining and electron microscopy. The cut is clean: all microtubules are severed and only a few extend beyond the others. This provides the basis for a clear test of whether traction fibers pull chromosomes to the pole in anaphase, because the putative traction fiber is cleanly severed. Cutting creates new plus ends on microtubules in the cut-off polar piece and new minus ends on microtubules in the main spindle body. The microtubules with new plus ends are unstable, as expected from the dynamic instability of microtubules. However, the microtubules with new minus ends are as stable as uncut microtubules in the same spindle. Our mechanical method of cutting microtubules very likely creates native, reactive ends, and therefore the surprising stability of new minus ends is genuinely interesting, not an artifact of cutting.
Replication occurs at discrete foci spaced throughout nuclei replicating in vitro
Authors: Mills A. D. , Blow J. J. , White J. G. , Amos W. B. , Wilcock D. , Laskey R. A.
Published: April 2021

Demembranated Xenopus sperm nuclei were induced to replicate synchronously in a low-speed supernatant (LSS) of Xenopus eggs by preincubation in a high-speed supernatant (HSS). DNA replication was observed by incorporation of [α-32P]dATP, BrdUTP or biotin-dUTP.

Biotin-dUTP incorporation, visualised with fluorescent streptavidin, reveals a striking pattern of replication foci throughout replicating nuclei. We show that this represents a precursor to the bright uniform fluorescence seen later. Confocal micro-scopic analysis of nuclei fixed early in replication reveals that these foci of DNA replication number about 100–300 for each nucleus and probably represent the replicon clusters already described for tissue culture cells. Foci are evenly distributed throughout the nuclei and are not concentrated at or near the nuclear envelope.

Complete replication of each nucleus occurs in an average time of only one hour in this system. Hence we calculate that there must be at least 300–1000 replication forks together in each cluster. Further-more, pulse labelling at later times in the period of replication reveals a similar pattern of foci indicating that replication forks remain tightly clustered in groups of at least 300 throughout the period of DNA replication.
Origin of type i collagen localized within oviduct epithelium of quail hyperstimulated by progesterone
Authors: Perche O. , Hayashi M. , Hayashi K. , Birk D. , Trelstad R. L. , Sandoz D.
Published: April 2021

Bird oviduct development is controled by sex steroid hormones. Estrogens (E) induce cell proliferation, formation of tubular glands by epithelial cell évagination and cell differentiation. Progesterone (P) strongly increases secretory processes in Etreated quails, but inhibits cell proliferation and cell évagination. The balance between E and P is very critical for the development and morphogenesis of the oviduct. After six daily injections of low doses of E (10 μg day−1) and high doses of P (5 μg day−1) into ovariectomized quails, cell proliferation and secretory process are stimulated but cell évagination is totaly inhibited and distribution of striated collagen is perturbed. Using antibodies against type I collagen the stroma, which is mainly composed of fibroblasts, is brightly stained, as are some regions within the epithelium. Electron microscopy shows that bundles of striated collagen fibrils appear in extracellular spaces between the lateral membranes of the epithelial cells or between the basal lamina and the epithelial basal membrane.

After in situ hybridization using a 35S riboprobe specific for mRNA of the α 2 chain of type I collagen, mRNA was detected only in the fibroblasts of the stroma and not in epithelial cells. Furthermore electron microscope studies of collagen bundles in serial sections clearly show collagen fibrils passing through the basal lamina. It is assumed that the type I collagen between epithelial cells originates from mesenchymal cells.

In the oviduct of immature birds or after physiological E+P stimulation, striated collagen is localized only in the stroma and never within the epithelium. These results indicate a modulation of extracellular matrix by sex steroid hormones in the quail oviduct.