bluuebunny/crossref_metadata_2025 huggingface dataset:
Conditions for fibroblast adhesion without fibronectin
Authors: Curtis A. S. G. , Mcmurray Heather
Published: April 2021

Conditions that permit the adhesion of BHK fibroblasts to a variety of surfaces after inhibition of protein synthesis and competition of any adsorbed fibronectin or vitronectin with the fibronectin cell-binding tetrapeptide, Arg-Gly-Asp-Ser (RGDS), are defined. Exposure of the cells to serum components at any stage in the preparation prevents cell attachment if cycloheximide or fibronectin tetrapeptide is present. If leupeptin is used cell adhesion and spreading occur even when all fibronectin synthesis is suppressed by cycloheximide inhibition, or fibronectin binding by tetrapeptide competition. The adhesions formed under these conditions appear by interferencereflection microscopy and by general properties to be identical to those formed by cells under normal culture conditions. The cell suspensions produced in the presence of leupeptin rather than other trypsin inhibitors show good adhesion at low temperatures, though the cells hardly spread at all. The results suggest that the role of fibronectin in cell adhesion should be reinterpreted in terms of its possible action as an activator rather than as a bonding molecule.
The role of the micronucleus in stomatogenesis in sexual reproduction of Paramecium tetraurelia: Laser microbeam irradiation of the micronucleus
Authors: Tam Lai-Wa , Ng Stephen F.
Published: April 2021

Fifteen amicronucleate cell lines and 22 cell lines with defective micronuclei were obtained following selective laser microbeam irradiation of the micronucleus. The amicronucleate cell lines showed reduced growth rate and formed abnormal oral apparatuses in asexual reproduction, and failed to produce any oral apparatus in autogamy, in agreement with previous observations on amicronucleate cells obtained by micropipetting. The 22 cell lines with defective micronucleus exhibited various abnormalities of the oral apparatus newly formed during autogamy. These abnormalities included the arrest of membranelle assembly, reduction in the length of the buccal cavity and oral membranelles, disruption of the organization of the membranelles, quadrulation of the dorsal peniculus, and failure of addition of membranellar basal body rows. Hence the micronucleus plays multiple roles in sexual stomatogenesis, deciding early steps of oral membranelle assembly and affecting their subsequent patterning. Our results agree with the notion that the micronucleus acts during a critical period between the second meiotic division and up to the formation of the zygotic nucleus to control the early stage of oral membranelle assembly. Laser microbeam irradiation might have created recessive mutations and/or chromosomal aberrations, which were expressed during this critical period with the formation of abnormal postmeiotic nuclei.
Affinity binding of non-histone chromatin proteins to the X chromosome of Drosophila by in situ chromatin reconstitution and its significance
Authors: Ukil Mitali , Chatterjee Kaberi , Dey Anup , Ghosh Saswati , Mukherjee A. S.
Published: April 2021

Cytophotometric analysis of the in situ binding affinity of non-histone chromosomal protein (NHCP) to the polytenic X chromosome and autosome of Drosophila melanogaster has been carried out using Feulgen-Napthol Yellow S staining technique. The results reveal that the mean transformed absorbance ratio (male:female) with a 547nm interference band filter for the two specific segments of the X chromosome is close to 0·5, while for a specific segment of an autosome it is close to 1·0, in the two sets of control; namely, the positive control (no treatment) and the negative control (treated with 1 M-urea+2M-NaCl) as well as in the reconstituted chromosomal preparations, which received 1 M-urea+2M-NaCl and the NHCP isolated from D. melanogaster. In contrast, the transformed absorbance ratios (male:female) with a 433 nm interference band filter yielded an interestingly different result. The ratios with a 433 nm filter for the X chromosome segments are significantly greater than 0·5 in all three sets of experiments. This finding by itself suggests that the NHCP binding affinity is dissimilar for the X chromosomes of male and female. When the 433 to 547 nm absorbance ratios were compared among the three sets, the data clearly revealed that in both positive control and NHCP reconstituted samples, the absorbance ratios (i.e. 433:547 nm) are significantly different between X chromosomes from males and those from females, while they are different between autosomes from males and females. The ratios are also not significantly different between male and female, either for the X chromosome or for the autosome in the negative control. These findings, therefore, suggest that there is a stronger binding affinity of NHCP for the male X chromosome of Drosophila, and reinstate the view that the X-chromosomal hyperactivity in male Drosophila is the consequence of a regulated organizational change in the DNA template.
Dissociation and reassociation of trichocyst proteins: Biochemical and ultrastructural studies
Authors: Peterson Joan B. , Heuser John E. , Nelson David L.
Published: April 2021

Trichocysts, the crystalline exocytotic organelles in Paramecium tetraurelia, are composed of small, acidic proteins existing primarily as disulphide-linked dimers. We have disaggregated trichocyst proteins with heat, simultaneously observing the changes in morphology and protein composition. The tip matrix was most heat-labile; its subunits progressively broke away from the distal end. During this process, breakdown of the cylindrical shaft began. Shafts first became flattened and torn lengthwise, yielding smaller, interconnected pieces still having the crystalline arrangement of their 5nm thick fibres. Ultimately this pattern became disordered, and discrete fibrils of the same thickness disengaged from the meshwork. In freeze-etched preparations these fibrils were composed of thinner filaments in side-by-side association. Disaggregation of the tip sheath began from the distal end before shaft dissociation was complete. Trichocysts broke down to thin fibrils, but probably not to monomeric subunits. At least three proteins were preferentially released in the initial phase of dissociation. Disulphide-reducing agent present during heating increased the rate of dissociation without altering the sequence of morphological changes or the order of release of individual proteins. The rate and extent of heat-induced dissociation were strongly dependent on pH and cation concentration. The stabilizing effects of low pH and of cations were additive.

A cooled suspension of fully dissociated trichocysts reassociated into sedimentable aggregates with discernible filamentous order, but without the crystalline structure of intact trichocysts. Reassociation was dependent upon time, temperature and protein concentration. All but one of the trichocyst proteins re-entered the sedimentable aggregate during reassociation.

Reassociation was faster and more complete at pH 6 than at pH 8 and was stimulated by Ca2+, Mg2+ and La3+. Trichocyst proteins dissociated in the presence of dithiothreitol did not reassociate, even after removal of the reducing agent.

Trichocysts from mutants defective to varying degrees in trichocyst formation were subjected to similar experimental protocols. Heat-dissociated trichocysts of the mutants scc6 and ptA1 reassociated at rates similar to those of wildtype; ftA3 showed slower reassociation, and tam38 showed little or no reassociation. Reassociation of wild-type trichocyst proteins was blocked by the addition of an equal amount of tam38 trichocyst proteins. Despite the variant structure, the main detectable difference in the protein complements of different strains was the relative prominence of common bands, rather than the presence of unique bands or the absence of common bands.
Effect of cell shape on proteinase secretion by epithelial cells
Authors: Hong H. L. , Brunette D. M.
Published: April 2021

Cell proliferation has been found to correlate with increased secretion of proteinases, such as plasminogen activator, in several different cell populations. In addition, the shape of the cell may also play a role in regulating proteinase secretion. However, the relationship between cell proliferation, cell shape and proteinase secretion has not been studied in diploid epithelial (E) cells cultured from porcine periodontal ligament (PL). We have modified PLE cell shape by physical means, such as growth on less-adhesive substrata and mechanical stretching, and by exposure to cholera toxin and 12-O-tetradecanoylphorbol-13-acetate (TPA). Neutral proteinase and plasminogen activator secretion were found to correlate with cell shape, the more round the cells, the greater the amount of proteinase secreted. PLE cells, stimulated to proliferate by cholera toxin or dibutyryl cyclic AMP, were more spread than control cells, but secreted less neutral proteinase and plasminogen activator. TPA stimulated cell proliferation slightly but, in contrast to cholera toxin, increased cell rounding and the secretion of neutral proteinase and plasminogen activator. Thus proteinase secretion was related more to cell shape than to cell proliferation.
Genotypic control of centromere positions of parental genomes in Hordeum × Secale hybrid metaphases
Authors: Schwarzacher-Robinson Trude , Finch R. A. , Smith J. B. , Bennett M. D.
Published: April 2021

The spatial disposition at metaphase of centromeres from Hordeum and Secale in root tip cells of H. chilense × S. africanum is described and compared with corresponding results for H. vulgare × S. africanum obtained previously. In both of these F1 sexual hybrids (2n = 2x = 14) each of the seven chromosome types from Secale was easily distinguished by its large size from any of the seven from Hordeum.

In H. chilense × S. africanum, centromeres of Secale chromosomes tended to be nearer the centre of the metaphase plate than did centromeres of Hordeum chromosomes in both squash preparations seen by light microscopy and unsquashed cells examined using electron microscope three-dimensional serial thin section reconstructions. This difference was significant in some individual cells, and highly significant for pooled data for reconstructed cells and separately for squashed metaphases. In no cell were Hordeum centromeres on average significantly nearer the centre of the metaphase plate than Secale centromeres.

These results agreed with those previously obtained for H. vulgare × S. africanum in that: (1) centromeres of the two parental haploid sets tended to be spatially separate; and (2) centromeres from one particular parent usually tended to be in the peripheral region of the metaphase plate that surrounded the central region containing the centromeres from the other parent. However, these results contrasted completely with those obtained previously in that Secale centromeres tended to be more central than Hordeum centromeres in H. chilense × S. africanum, but more peripheral than Hordeum centromeres in H. vulgare × S. africanum.

As centromeres of the parental set with the larger chromosomes (i.e. Secale) can be either inside, or outside, centromeres from the parental genome with the smaller chromosomes (i.e. Hordeum), then clearly, a tendency for a concentric separation of parental genomes is not a packing phenomenon determined by chromosome size perse, but is presumably under genotypic control.
Association of calmodulin with lysosomes
Authors: Nielsen Thor B. , Field James B. , Dedman John R.
Published: April 2021

We examined the subcellular localization of calmodulin in several cultured cells (primary thyroid follicular cells, thyroid C-cell tumour cells (TT), kidney cells (PtK2-L23) and peritoneal macrophages) by indirect immunofluorescence using affinity-purified antibody to calmodulin. When cells were fixed with 3 % formaldehyde for 15 min prior to lysis with 0·5% Triton X-100, spindle fibres in mitotic cells were fluorescent and a diffuse cytoplasmic localization of calmodulin was observed in resting cells. However, when cells were lysed with 0·5% Triton X-100 for 90s prior to fixation for 30min with 3% formaldehyde, three effects were observed. One: there was little diffuse cytoplasmic staining. Two: discrete vesicles were stained. Three: spindle fibres in mitotic cells were fluorescent. The stained vesicles were phase-dense and ranged from 0·1 to 0·5μm in primary thyroid follicular cells but were smaller in PtK2 and TT cells. The thyroid follicular cells retained vesicular staining after exposure to thyrotropin and isobutyl-methylxanthine, but the number of labelled vesicles decreased by almost 80%. Phase-dense vesicles were identified as lysosomes or other acidic vesicles by vital staining with Acridine Orange. After differential centrifugation of thyroid homogenates, calmodulin was measured by radioimmunoassay (RIA) and found in both cytosolic (89 %) and membrane vesicle fractions (11 %). The vesicular calmodulin was not eluted by washing with 5 mM-EGTA. The thyroid fractions were subjected to SDS–polyacrylamide gel electrophoresis and the gels incubated with 125I-labelled calmodulin to reveal calmodulin acceptor proteins (CAPs). The vesicle fraction contained quantitatively major CAPs with Mr of 200000, 140000, 89000, 38000 and 34000, and minor CAPs of 60 000 and 50 000. Washing the pellet with 5 mM-EGTA did not reduce the content of CAPs. Thus, calmodulin and CAPs are present both in the cytoplasm and in a membrane vesicle fraction. The lysosomal locale of calmodulin and the effect of thyrotropin on vesicle number suggest a role for calcium in the regulation of lysosome function.
The suppression of malignancy by terminal differentiation: Evidence from hybrids between tumour cells and keratinocytes
Authors: Harris Henry , Bramwell Michael E.
Published: April 2021

When malignant human cells are crossed with diploid human keratinocytes, malignancy, as defined by progressive growth in vivo, is suppressed so long as the hybrid cells continue to produce involucrin, a protein that characterizes terminal differentiation in the kératinocyte. When, on continued cultivation in vitro, the cells lose the ability to produce involucrin, they reacquire the ability to grow progressively in the animal.
Dissection of the bovine epidermal desmosome into cytoplasmic protein and membrane glycoprotein domains
Authors: Skerrow Christine J. , Hunter Irene , Skerrow David
Published: April 2021

Epidermal desmosomes contain two main regions. The core consists of a pair of membranes, one on either side of a cross-striated intercellular space bisected by a denser midline. The cytoplasmic compartment comprises a dense plaque deposited on the cytoplasmic surface of each membrane and a diffuse layer occupying the zone between the plaque and attached cr-keratin filaments. Analysis of isolated desmosomes by SDS-PAGE has shown the presence of four major protein (dpl-4) and three major glycoprotein (dgl-3) bands, which have been allocated to the cytoplasmic and core compartments, respectively.

In the present paper, we report the use of urea to fractionate this complex structure, both in situ and following isolation with citrate buffer, pH2’6. Extraction of the living layers of bovine epidermis with 9M-urea, pH 7 · 5, resulted in rapid removal of the dense desmosomal plaques, followed by separation and vésiculation of desmosomal membranes. The resistance of the plaque to urea increased abruptly at the transition between living epidermis and dead, dehydrated horny layer. A similar sequence of morphological changes accompanied the extraction of isolated desmosomes with urea. Analysis of residues and extracts of isolated desmosomes by SDS-PAGE confirmed the selectivity of 9M-urea, pH 7 · 5, for the cytoplasmic compartment. The four major desmosomal proteins, dpl-4 (Mr240, 215, 90 and 83 (× 103), respectively) predominated in the extracts. Desmosomal membranes, both paired and vesiculated, consisted almost entirely of the three desmosomal glycoproteins dgl-3 (Mr 150, 120 and 110 (× 103), respectively).

These results provide evidence that all three desmosomal glycoproteins are integral membrane proteins. The separation of desmosomal membranes by urea, which is not accompanied by additional loss of proteins, further suggests that desmosomal adhesion is based on interactions between membrane components with no separate extracellular molecules being involved. The dissection of the desmosome by urea into two topographically and biochemically distinct domains should facilitate further studies on the molecular basis of desmosomal adhesion and a- keratin filament binding.
Characterization of Pales spermatocyte spindles, with reference to an mtoc-associated protein
Authors: Bastmeyer Martin , Russell David G.
Published: April 2021

Pales ferruginea spermatocytes, because of their high proportion of dividing cells, offer many advantages in the study of the meiotic spindle. In the present study we have combined ultrastructural and immunofluorescence techniques with biochemical analysis of constituent spindle components. Using a centrosome-reactive serum, which recognizes an antigen in the centrosomal and midbody regions of dividing cells, we have identified an MTOC-associated polypeptide of 112000Mr with a pl of 5·6. The relative abundance of this protein in cells and isolated cytoskeletons indicates that it is present in other regions of the cell and is either more abundant, or is only recognized by the antibody in association with MTOCs.
Regional differentiation of the membrane skeleton in Tetrahymena
Authors: Williams N. E. , Honts J. E. , Jaeckel-Williams R. F.
Published: April 2021

Antisera have been raised in rabbits against three high molecular weight proteins that are present in Triton X-100-insoluble residues of Tetrahymena pyriformis GL cells. These proteins, called A, B and C, have apparent molecular weights of 235, 135 and 125 (×103), respectively, in SDS-polyacrylamide gels. The antisera obtained are specific for these proteins, as shown by immunoblotting. Immunolocalization studies are reported that suggest that these proteins are present throughout the epiplasmic layer beneath the cell surface (membrane skeleton). Images obtained with the fluorescence microscope, however, suggest that the membrane skeleton is modified in discrete zones: (1) around somatic basal bodies, (2) within the oral apparatus, (3) in the cytoproct, (4) in contractile vacuole pores, (5) in the fission zone in late division, and (6) at the mating junction in conjugating cells. These regions may represent areas of increased rigidity at the cell surface. The transition from pliable to rigid epiplasm in spatially delimited areas is apparently a recurring theme in cortical morphogenesis in Tetrahymena. Together, the two types of epiplasm probably allow for extensive changes in cell shape while preserving essential relationships between structural elements within the cortex.
Newt epidermal cell migration in vitro and in vivo appears to involve Arg-Gly-Asp-Ser receptors
Authors: Donaldson Donald J. , Mahan James T. , Smith Gerald N.
Published: April 2021

The effect of a synthetic peptide consisting of Arg-Gly-Asp-Ser (RGDS), the amino acid sequence representing the fibroblast attachment site in fibronectin (FN), was tested on migrating newt epidermal cells. In one approach, skin explants were placed on the bottom of plastic dishes coated with human FN, human fibrinogen (FGN), human serum spreading factor (SF), or bovine type I collagen. The explants were then incubated overnight in serum-free medium with or without RGDS. In these experiments exposure to 50 μgml−1 of RGDS reduced migration over FN, FGN and SF to 2–7% of control levels. Two peptides structurally dissimilar to RGDS (Vai-Gly-Ser-Glu and Thr-Pro-Arg-Lys), and two that are structurally similar (Lys-Gly-Asp-Ser and Arg-Gly-Glu-Ser), had no effect on explant migration even when used at concentrations higher than 50 μgml−1. Upon removal of the RGDS peptide, inhibited explants quickly recovered. In collagen-coated dishes 50 μgml−1 of RGDS was much less effective than in dishes coated with the other substrates. Raising the RGDS concentration in collagen-coated dishes tenfold did not greatly increase the RGDS effect. When added to the medium bathing wounded limbs, 50 μg ml−1 of RGDS only moderately inhibited wound closure.

This concentration of peptide, however, severely inhibited migration from skin explants in newt-plasma-coated-dishes and migration over pieces of newt-plasma-coated plastic placed under one edge of a skin wound. Increasing the RGDS concentration to 500 μg ml−1 resulted in almost total suppression of wound closure. Wounds exposed to this same concentration of Lys-Gly-Asp-Ser closed normally.

These results indicate that newt epidermal cells possess RGDS receptors and that these receptors are involved in epidermal wound closure in vivo and in migration from skin explants onto plastic coated with FN, FGN, SF and collagen.

The relative RGDS-insensitivity of wound closure in vivo and in migration from explants onto collagen may reflect in these instances the presence of a relatively high density of RGDS receptor binding sites on the substrate; the presence of RGDS receptor binding sites of relatively high affinity; or the participation of receptors other than those involved in migration over plastic coated with FN, FGN or SF.
Visualization of the intact endoplasmic reticulum by immunofluorescence with antibodies to the major er glycoprotein, endoplasmin
Authors: Koch G. L. E. , Macer D. R. J. , Smith M. J.
Published: April 2021

Antibodies to endoplasmin were used to examine the morphology of the endoplasmic reticulum (ER) by immunofluorescence on permeabilized plasmacytoma and fibroblastoid cells. In unfixed cells, permeabilization led to a pronounced vésiculation of the ER. Therefore cells were first fixed lightly prior to permeabilization with detergent. Fibroblastoid cells gave a characteristic reticular pattern surrounding the nucleus with clear staining of the nuclear membrane. Plasmacytoma cells, in the conventional fluorescence microscope, gave a cistemae-like pattern. Optical sectioning with a confocal scanning laser microscope gave a distinct pattern of concentric cisternae similar to those obtained with transmission electron microscopy on cell sections. The overall morphology of the ER in such cells could be revealed by serial optical sectioning. Evidence was obtained that the ER does not undergo extensive vésiculation during mitosis in plasmacytoma cells. Using anti-endoplasmin immunofluorescence monitoring, conditions were developed for the retention of ER morphology in unfixed, permeabilized cells.

These studies illustrate the value of endoplasmin as a general marker for the analysis of ER morphology in different types of cells by immunofluorescence microscopy.
Interaction In Vitro Of Non-Epithelial Intermediate Filament Proteins With Supercoiled Plasmid Dna*
Authors: KÜhn S. , Vorgias C. E. , Traub P.
Published: April 2021

Sucrose gradient analysis of reaction products obtained from non-epithelial intermediate filament (IF) subunit proteins and a mixture of supercoiled, relaxed and linearized plasmid pBR322 DNA at low ionic strength revealed that limited amounts of these polypeptides interacted exclusively with the supercoiled form of the plasmid DNA. These results were corroborated by electron-microscopic analysis of the reaction products, which showed that only circles of supercoiled pBR322 DNA were completely and smoothly covered with vimentin. LFs reconstituted from pure vimentin reacted with supercoiled pBR322 DNA only through their physical ends. The reaction of an aged preparation of vimentin with supercoiled pBR322 DNA produced large aggregates consisting of a central, axially oriented protein scaffold to which individual loops of DNA were attached at their bases in a halo-like arrangement. The electron-microscopic appearance of such complexes was very reminiscent of that of histone-depleted metaphase chromosomes. Together with the previous observations that non-epithelial IF proteins have high affinities for single-stranded DNA and core histones and that they are structurally and functionally closely related to the nuclear lamins, these results were used to advance a novel hypothesis on the biological role of IF proteins in eukaryotic cells.
Polarized Pigment Granule Transport Occurs In The Absence Of Microtubules In Squirrelfish Erythrophores: Studies Of The Effects Of Estramustine
Authors: Stearns Mark E. , Wang Min
Published: April 2021

We have re-examined the involvement of microtubules in the process of pigment granule transport in squirrelfish erythrophores in situ (i.e. on scales). Light-microscopic studies revealed that following exposure to 5 μM-nocodazole for 1 h at 4 °C erythrophores retained an ability to aggregate and disperse their pigment uniformly, though at reduced rates. Serial thick-section stereo high-voltage electron-microscopic studies showed that the entire microtubule population was removed by drug treatment and that the microtubules were not reassembled as a result of pigment translocation processes in the presence of reduced levels of nocodazole (0–4 μM). Immunofluorescence microscopic studies confirmed that nocodazole (0’5–1 μM) produced rapid disassembly of the microtubules. Wholemount electron-microscopic studies showed that the pigment granules were suspended in a crosslinking network of 3–10nm filaments, which appeared to support ordered pigment transport in situ in the absence of microtubules. Drug inhibition studies showed that micromolar levels of estramustine, a novel anti-MAPs (microtubule-associated proteins) drug, reversibly inhibited pigment transport. The results suggest that an estramustine-sensitive cytomatrix component might produce polarized pigment transport in intact erythrophores.
A major difference between serum and fibronectin in the divalent cation requirement for adhesion and spreading of BHK21 cells
Authors: Edwards J. G. , Robson R. T. , Campbell G.
Published: April 2021

Adhesion and spreading of BHK21 cells on adsorbed bovine and foetal bovine serum require addition to the medium of a divalent cation. Divalent cations are effective in the order Mn2+>Co2+>Mg2+>Ca2+, with Ca2+ ineffective below 10−4M. On purified fibronectin, however, no added divalent cation is required, since the requirement is largely met by adventitious Ca2+ (circa 10−5M) in nominally divalent cation-free saline. In such background Ca2+, adhesion and spreading on fibronectin are only slightly slower than in optimal Mg2+, and appear identical, morphologically, and in sensitivity to cytochalasin D. Cells also spread on fibronectin in response to Mg2+, when Ca2+ is buffered below 10−6M, showing that external Ca2+ is not needed as a source for an increase in internal Ca2+. Cells can be induced to spread on serum in low Ca2+ by substantially increasing the fibronectin concentration; this supports other evidence that at its unsupplemented concentration, fibronectin contributes little to the spreading of these cells on serum.

The Ca2+ requirement for spreading on preparations of vitronectin (serum spreading factor), partially purified from bovine serum, is similar to that on whole serum. Thus the difference in divalent cation requirement between serum and fibronectin may arise because, on serum, the dominant protein responsible for induction of spreading is vitronectin rather than fibronectin. We argue that such a difference in ion requirement between these surfaces points to a site of action of the ions at the cell surface, perhaps directly on binding of the adsorbed proteins by their receptors, or in receptor-specific transduction events, rather than via ion fluxes, on cyto-skeletal responses common to different surfaces.

Models are discussed to account for why the divalent cation requirement should differ for interaction of cells with different substrate-adsorbed proteins.
Establishment of a tissue culture system for epithelial cells derived from human pancreas: a model for the study of cystic fibrosis
Authors: Harris Ann , Coleman Lindsay
Published: April 2021

A tissue culture system for epithelial cells derived from human foetal pancreas has been established. The cultured cells show many ultrastructural features of interlobular duct cells. Immunocytochemical and histochemical evidence is presented in support of the view that these cells are ductal in origin. They are likely to be one of the few cell types that express the basic defect of cystic fibrosis in vitro. The cells may be passaged and sufficient material obtained to permit biochemical and molecular biological analysis.
An endothelial cell growth factor derived from human lung carcinoma cells grown in serum-free medium
Authors: Walker C. , Mates G. , Pumford D. , Daniel M.
Published: April 2021

A factor that stimulates the proliferation of human umbilical vein endothelial cells has been shown to be present in serum-free medium conditioned by the prior growth of a cell line (1PT) derived from a poorly differentiated bronchial carcinoma. Preliminary characterization of this factor has revealed that it is a heat-labile, acidstable proteinaceous material, the activity of which is not diminished by treatment with a reducing agent. In its partially purified state ithas been shown to be anionic and to be associated with material exhibiting a broad molecular weight range of 35×103 to 100×103. It does not bind strongly to heparin-Sepharose and its mitogenic effect on endothelial cells is not potentiated by heparin. These properties suggest that this factor may differ from other previously described tumour-derived endothelial mitogens.
Cell Movement and Cell Behaviour
Authors: Dunn G. A.
Published: April 2021

John Lackie’s course on cell behaviour delivered to Senior Honours Cell Biology students at Glasgow University is largely responsible for the predominance of young Glaswegian scientists at British conferences on cell behaviour. Other universities have an oppor tunity to redress the balance now that this lecture course is available as a textbook. But this book, particularly the latter part, is so original in its treatment of the field that it deserves to be read by anyone with even a passing interest in the subject. It is not the first textbook to deal with cell behaviour. Others deal with the motile behaviour of embryonic cells during development, with the chemotaxis of white blood cells or with the invasiveness of malignant cells. But it is the first attempt to present a coherent survey of the whole field of cell behaviour with the emphasis on common principles. It does mainly for the metazoan cell what the now classic textbook, The Orientation of Animals by Fraenkel & Gunn (1940), did mainly for the protozoa and other small animals.
Clonal lifespan of Paramecium tetraurelia: Effect of selection on its extension and use of fissions for its determination
Authors: Takagi Yoshiomi , Nobuoka Tomoko , Doi Mayumi
Published: April 2021

Paramecia cells, like human diploid cells cultured in vitro, provide a useful model system for understanding the mechanism that limits division potential. The reported maxima of the clonal lifespan of Paramecium tetraurelia fall into two ranges: from 220 to 258 fissions and from 310 to 325 fissions. We found that neither the selection of vigorous lines nor the cryptic occurrence of autogamy offers a plausible explanation for the much longer lifespans in the latter range. We found the sporadic occurrence of very long clonal lifespans, such as 330 fissions, without selection and autogamy. Selection, which was evaluated by using different methods to maintain lines, had little effect on the extension of the maximal clonal lifespan, whereas it did have a marked effect on the extension of the mean clonal lifespan. Autogamy, which was checked with two closely linked marker genes, was frequent, but only during the period when lines were terminating.

Statistical analysis on the mean clonal lifespans of two groups of subclones cultured at 25 and 20 °C or with rich and poor nutrition showed that the clonal lifespan that was fission-related under favourable conditions tended to dissociate from fissions under less favourable conditions. We discuss mechanisms that determine the clonal lifespan, programmed events contributing to the maximum clonal lifespan and random events contributing to the mean clonal lifespan.
Lampbrush chromosomes
Authors: Macgregor H. C.
Published: April 2021

1986 was a celebration year for lampbrush chromosomes (LBCs), marked by the publication of Callan’s comprehensive and authoritative book on these structures. My commentary begins where Callan’s book ends, standing on tiptoe beside a large and rather neat assembly of well-established facts, principles and hypotheses, and trying hard to catch a glimpse of what lies ahead.
Calcium control of macrophage cytoplasmic gelation: Evidence for the involvement of the 70000Mr actin-bundling protein
Authors: Pacaud M. , Harricane M. C.
Published: April 2021

Under appropriate conditions macrophage cytosolic extracts can form a three-dimensional gel network of cross-linked actin filaments. These cytoplasmic gels are mainly composed of actin, filamin, alpha-actinin, and two new proteins of about 70000 and 55 000Mr (70 and 55 K). The behaviour of 70 K protein was found to be remarkably affected by Ca2+. Ca2+ treatment of isolated cytoplasmic gels led to the selective solubilization of the 70 K protein along with a 17 K polypeptide. Half-maximal recovery in the supernatant fraction was obtained from about 0·15μM free Ca2+. The cytoplasmic gel constituents solubilized in high ionic strength buffer were able to re-assemble into an insoluble actin network when returned to near physiological ionic conditions. However, the inclusion of micromolar Ca2+ prevented the re-association of 70 K protein with actin in these complexes. As compared to the 70 K protein, alpha-actinin was fully resistant to any variations in Ca2+ concentrations. On the other hand, purified 70 K protein displayed the property of assembling actin filaments into bundles at low Ca2+ concentrations (<0·15 μ M). However, the bundling activity decreased progressively at higher Ca2+, as detected by co-sedimentation and electron microscopy of the 70 K protein-actin mixtures. Half-maximal inhibition was observed at about 0·3 μM free Ca2+. Re-assembly of actin filaments into bundles occurred after chelation of Ca2+ by EGTA, indicating that the inhibitory effect of Ca2+ was reversible. Severing of actin filaments by 70 K protein was not observed in any of the solution conditions used.

The Ca2+-dependent inhibition of the ability of 70 K protein to interact with actin networks resulted in a marked distortion of the overall organization of actin filaments, as revealed by thin-section electron microscopy of cytoplasmic gels formed in the presence and absence of Ca2+. Large zones of oriented bundles of filaments were replaced by a looser mesh. When the actin gel constituents were re-assembled in the presence of Ca2+ and exogenous gelsolin, it was also observed that the filament bundles (essentially generated by alpha-actinin) collapsed into dense aggregates. Furthermore, gelsolin did not significantly affect the ability of actin to re-combine with other proteins. The data presented here and previously led us to suspect that the Ca2+ control of the functional state of 70 K protein might be one of the prime factors in the triggering of rapid assembly and disassembly of microfilaments within macrophages.
Evidence for microtubule nucleation at plasma membrane-associated sites in Drosophila
Authors: Mogensen M. M. , Tucker J. B.
Published: April 2021

This report is concerned with the nucleation and organization of microtubule bundles that assemble after ‘conventional’ centrosomal microtubule-organizing centres have been lost. The microtubule bundles in question span the lengths of wing epidermal cells. Bundles extend between hemidesmosomes at the apical cuticlesecreting surfaces of cells and basal attachment desmosomes that unite the dorsal and ventral epidermal layers of developing wing blades. Furthermore, each bundle includes up to 1500 microtubules and most of the microtubules are composed of 15 protofilaments.

Individual cells were serially cross-sectioned at an early stage of bundle assembly. The number of microtubule profiles/cell cross-section decreased progressively by up to 59% of the most apical values in section sequences cut from fairly apical to more basal levels in the cells. The apical ends of microtubules were associated with numerous small dense plaque-like sites (diameter 0·1–0·2 gm), which were specialized regions of plasma membranes at the apical surfaces of cells. Many of the microtubules near apical plaques were not well aligned with each other; they ‘radiated away’ from cell apices. This was in contrast to the situation at more basal levels where most microtubules were oriented parallel to the longitudinal axes of cells.

These findings indicate that the relatively dispersed arrays of apical plasma membrane-associated plaques act as microtubule-nucleating sites to initiate basally directed elongation of bundle microtubules. Apical cell surfaces and their plaques seem to operate as microtubulenucleating and -organizing regions that functionally replace the centrosomal microtubule-organizing centres lost earlier in cell differentiation.
The neuron as a cell
Authors: Amos L. A.
Published: April 2021

The neuron is a particularly useful type of cell for studying many general processes. The site of locomotion, the growth cone, is well-removed from most of the cytoplasm; thus it is possible to exclude direct involvement in locomotion for many cytoplasmic components and less of a problem to identify those that are important. The spatial separation between sites of synthesis or reprocessing in the cell body and the terminal membrane also helps in resolving the pathways of membrane-associated proteins that are recycled, degraded or exported. And, of course, the long distances that substances must travel along an axon permit studies of intracellular transport mechanisms. This volume, the proceedings of a Satellite Symposium of the 30th Congress of the International Union of Physiological Sciences, held a year ago at the University of Calgary, Alberta, brings together these various topics. A series of papers by different researchers provides much to interest cell and molecular biologists in general, as well as those scientists who are interested in the nervous system per se.
Ganglioside inhibition of attachment and differentiation of cultured rat granulosa cells: interactions with fibronectin
Authors: Morley Paul , Armstrong David T. , Gore-Langton Robert E.
Published: April 2021

The involvement of fibronectin in the attachment and differentiation of rat granulosa cells, cultured in a chemically defined medium, was investigated using the inhibitory properties of mixed brain gangliosides (MBGs) and highly purified disialoganglioside, GD1a. MBGs inhibited cell attachment to plastic culture surfaces in a concentration-dependent manner, with 0·1 mmol 1-1 causing significantly decreased attachment between 0·5 and 24 h of incubation. Inhibition of attachment to a fibronectin-coated substratum was even greater. The inhibitory effect of MBGs was not caused by binding to the cell surface, but instead the inhibitory factor(s) were adsorbed on a surface of immobilized human plasma fibronectin, thereby preventing cell attachment to this surface. The inhibitory action of MBGs was also neutralized by the addition of soluble fibronectin. Furthermore, at least one component of MBGs, detected chemically following thin-layer chromatography, was directly shown to bind to human fibronectin.

MBGs inhibited to varying degrees the folliclestimulating hormone(FSH)-dependent responses: augmentation of cellular protein content, production of adenosine 3′,5′-cyclic monophosphate (cyclic AMP) and progestins (progesterone-1-20cv-hydroxypregn-4-en-3-one+pregnenolone), and induction of aromatase activity. These inhibitory activities of MBGs could not be eliminated by adsorption on immobilized fibronectin or reversed by addition of soluble fibronectin, thus distinguishing these actions from the early inhibition of cell attachment. FSH-dependent responses were also inhibited by GD1a, while responses to stimulation by dibutyryl cyclic AMP plus 3-isobutyl-l-methyl xanthine were less affected by this ganglioside.

These results suggest that gangliosides inhibit attachment of granulosa cells in culture by binding to fibronectin, whereas the inhibition of FSH-dependent differentiation occurs by other modes of action that are unrelated to the effects on cell adhesion.
The permeability properties of septate junctions in Malpighian tubules of Rhodnius
Authors: Le H. , Skaer B. , Maddrell S. H. P. , Harrison J. B.
Published: April 2021

This paper describes the structural characteristics and permeability properties of the smooth septate junctions between the upper Malpighian tubule cells of a blood-sucking bug, Rhodnius prolixus. The permeability of the paracellular route was tested only for solutes that could be demonstrated not to cross the epithelium via the cellular route. The intercellular clefts were readily permeated by sucrose, inulin and polyethylene glycol (PEG), showing a higher permeability to molecules of smaller radius (PEG versus sucrose). Negatively charged molecules permeated the clefts more readily than positively charged ones. The effects of pH, urea and luminal flow rate on permeability were studied. The results are discussed in relation to the physiological tightness of the Malpighian tubules to certain solutes and to its function as an excretory epithelium.
Strategies and mechanisms for the control of transcriptional initiation of mammalian protein-coding genes
Authors: Melton David W.
Published: April 2021

An ability to exercise precise control over a large number of genes, with widely differing patterns of expression, is a requirement for all organisms and this ability reaches its most sophisticated level in mammalian systems. Although there are numerous examples of control being exerted at every stage along the expression pathway, the key control point in most instances appears to be at the first stage of the process - the transcriptional initiation event itself. The purpose of this article is to provide a brief overview of current ideas on the control of transcriptional initiation in mammalian cells, both in the context of recent developments in mammalian systems and by analogy with more advanced studies in prokaryotes and yeast. (See Ptashne, 1986; Struhl, 1987, for reviews on transcriptional initiation in bacteria and yeast, respectively.)
Structure of the generative cell wall complex after freeze substitution in pollen tubes of Nicotiana and Impatiens
Authors: Cresti M. , Lancelle S. A. , Hepler P. K.
Published: April 2021

The mature generative cell in pollen grains and pollen tubes is surrounded by a wall complex that includes two plasma membranes, one facing the generative cell cytoplasm and one facing the vegetative cell cytoplasm, and usually some intervening wall material. After conventional chemical fixation, the two plasma membranes are very uneven and often appear to be joined, giving the impression that numerous plasmodesmata connect the vegetative and generative cells. These areas alternate with swollen, distorted areas, which give the wall complex the appearance of being composed of a chain of vesicles. Utilizing rapid freeze fixation and freeze substitution, we have re-examined the ultrastructure of the generative cell wall complex from pollen tubes grown in vitro, and the differences are striking. The two plasma membranes are very smooth and closely appressed to a layer of wall material. Occasionally the wall complex contains swollen areas, or varicosities, and these may contain pockets of lightly stained material, but again the surrounding plasma membranes are tightly appressed to these areas. Plasmodesmata are not seen, but this does not eliminate the possibility that they may exist at an earlier stage of development.
Effects of calcium antagonists on cyclic AMP phosphodiesterase induction in Dictyostelium discoideum
Authors: Coukell M. B. , Cameron A. M.
Published: April 2021

Previous studies have suggested that cyclic GMP and/or Ca2+ might function as second messengers in the induction by exogenous cyclic AMP of the cyclic AMP phosphodiesterase (PD) in Dictyostelium discoideum. To assess further the role of Ca2+ in PD induction we examined the effect on this process of a number of putative Ca2+-channel blockers. At relatively low micromolar concentrations, TMB-8, nicardipine, nifedipine, diltiazem and verapamil all altered cell shape and inhibited PD induction in a similar dose-dependent fashion. Concentrations of these drugs that abolished PD induction had no effect on cell viability; however, higher concentrations reduced viability and caused cell lysis. All effects of these compounds on the cells were antagonized at least partially by 5–10mM-Ca2+. Other cations tested were considerably less effective. Like the organic inhibitors, La3+ also altered cell shape, inhibited PD induction and reduced cell viability at elevated concentrations, but its effect on the cells appeared to be more complex. Inhibition of PD induction by the organic antagonists could not be, attributed solely to an impaired uptake of extracellular Ca2+, a reduction of ATP pools in the cells or a direct effect on calmodulin. Concentrations of TMB-8 that inhibited PD induction had little effect on the cyclic GMP response. Therefore, this compound did not inhibit PD induction indirectly by blocking cyclic GMP synthesis. While the sites of action of these drugs could not be determined precisely, the results are consistent with the hypothesis that intracellular Caz+ functions as a regulator of PD induction in this organism.
adaptation to chemotactic cyclic amp signals in Dictyostelium involves the g-protein
Authors: Small Nadine V. , Europe-Finner G. Nicholas , Newell Peter C.
Published: April 2021

Amoebae of Dictyostelium discoideum show adaptation towards a chemotactic cyclic AMP signal. Within a few seconds of receipt of the signal they are inhibited for a period of 1–2 min from further chemotactic responses to subsequent cyclic AMP signals of similar or smaller magnitude. The site of this adaptation mechanism in the chemotactic transduction pathway was investigated by addition of components of the transduction chain (GTP analogues, myo-inositol-1,4,5-trisphosphate (InsP3) and Ca2+) to permeabilized cells followed by determination of the amount of cyclic GMP formed as a measure of the chemotactic response. This approach was made possible by finding that permeabilization of amoebae with saponin did not uncouple the cell surface cyclic AMP receptors from stimulation of cyclic GMP formation.

It was found that InsP3 and Ca2+ were ‘downstream’ from the adaptation mechanism: they could trigger a cyclic GMP response in cyclic AMP-adapted amoebae but could not themselves induce adaptation. In contrast, GTPγS was unable to trigger a cyclic GMP response in cyclic AMP-adapted cells, although it could trigger multiple cyclic GMP responses in non-adapted cells. We deduce that the site of adaptation to cyclic AMP stimulation is at the G-protein involved in this signalling pathway. Moreover, as GTPγS was found to be unable to induce adaptation, we conclude that the mechanism of adaptation involves an action of the cyclic AMP receptor on the G-protein that is distinct from its commonly reported action of stimulating G-protein binding of GTP.