bluuebunny/crossref_metadata_2025 huggingface dataset:
Macromolecular glucosamine-containing component of the surface of cultivated mouse cells
Authors: Onodera K. , Sheinin Rose
Published: April 2021

It has been demonstrated that a glucosamine-containing macromolecular component of the cell surface of 3T3 mouse cells, and SV4O-transformed cells, is released from cells by treatment with trypsin under conditions in which the plasma membrane remains functionally intact. This was shown by the fact that the treated cells could be cloned with high plating efficiency and remained impermeable to the vital stain, erythrocin. A method for specifically marking this surface component has been devised based on the finding that in 3T3 cells growing synchronously after subculture by trypsin maximum incorporation of glucosamine into this material occurs 12 − 13 h thereafter. Of the total radioactive glucosamine incorporated into macro molecular cell constituents, over 80 % was recovered in surface component.

Studies on the biosynthesis of surface component revealed that this was periodic during a cycle of cell duplication, with an increased rate of formation immediately after cell division. It was found that the surface component of 3T3 cells differed from that of SV40-transformed cells.
Control of flagellum number in naegleria : temperature shock induction of multiflagellate cells
Authors: Dingle A. D.
Published: April 2021

This paper describes the production of supernumerary flagella by flagellates of Naegleria gniberi exposed to sublethal temperature shocks during the amoeba-to-flagellate transformation. When transformed at any constant temperature below 34°C, cells of N. gniberi strain NB-1 may develop from 1 to 4 flagella, but biflagellate cells predominate and the average number of flagella per cell in these populations is approximately 2 ·2. Populations exposed to a 38 °C temperature shock during transformation develop about twice as many flagella as controls (average number of flagella per cell is approximately 4·5). The individual response of cells is extremely heterogeneous: some develop no more than the normal 2 flagella, whereas others develop as many as 18. At least half the cells produce 5 or more flagella, as opposed to fewer than 1 % with 5 or more flagella in control populations. Multiflagellate cells and populations are normal in appearance, apart from the excess flagella and resulting disorientation of the normal swimming pattern. The supernumerary flagella, their basal bodies and the rhizoplast (which is also occasionally doubled) are indistinguishable from those of normal cells. Maximal production of flagella occurs over the narrow temperature range from 37 · 5 to 38 ·5 °C and is related to the duration of exposure to a given temperature: optimal flagellum induction is obtained following a 45 · 50 min exposure to a 38 · 2 °C temperature shock. After approximately 1 h the flagellates revert to amoebae, losing both normal and supernumerary flagella. Populations of these amoebae followed through 2 more cycles of retransformation and reversion developed the normal number of flagella (2 · 2). Clones isolated from multiflagellate populations were identical to normal populations, developing approximately 2 · 1 flagella per flagellate at normal temper atures and 4 ·5 flagella per flagellate when temperature shocked again.
The effect of 2-phenyl ethanol on the dna synthesis cycle of Schizosaccharomyces pombe
Authors: Bostock C. J.
Published: April 2021

The effect of different concentrations of 2-phenyl ethanol (PE) on growth and DNA synthesis of Schizosaccharomyces pombe is described. 0·3 % PE inhibits the entry of cells into 5 phase, but allows a doubling in the number of cells in the culture. The effect of 0·2 % PE on random and synchronous cultures of S. pombe shows that, in the continued presence of the inhibitor, the S phase is moved to a different point in the cell cycle. Cells continue to grow in the presence of 0· 2 % PE with a G1 phase occupying a significant portion of the cell cycle. This differs from normal growth when the G1 phase is absent.
The competitive effects of serum proteins on cell adhesion
Authors: Curtis A. S. G. , Forrester J. V.
Published: April 2021

Binding curves for the adsorption of plasma fibronectin, alpha-l-antitrypsin, alpha-2-macroglobulin, ceruloplasmin, transferrin and bovine serum albumin to plain and to hydroxylated polystyrene surfaces were measured. These curves were correlated with the adhesion of BHK cells and leucocytes to these adsorbed protein surfaces in protein-free culture media. Hydroxylated polystyrene adsorbed less of alpha-l-antitrypsin, alpha-2-macroglobulin and albumin than the plain polystyrene. On the other hand the hydroxylated surfaces bound more fibronectin than the plain polystyrene surfaces. Hydroxylated polystyrene surfaces were also more adhesive for both BHK cells and leucocytes than plain polystyrene: a result confirming earlier work. The competition of fibronectin for adsorption to plain polystyrene with alpha-l-antitrypsin, alpha-2-macroglobulin and ceruloplasmin was measured and correlated with effects on cell adhesion. The results suggest that the low adhesiveness of BHK cells and leucocytes on plain polystyrene in sera-containing media is due both to the low binding of fibronectin and to the binding of serum albumin, alpha-l-antitrypsin and alpha-2-macroglobulin. The relative unimportance of fibronectin in adhesion to these surfaces is shown by the finding that cell attachment will not occur to polystyrene surfaces that have bound high levels of the antiadhesive proteins in the presence of fibronectin, even though attachment will occur in the absence of fibronectin provided that the antiadhesive proteins are lacking.
An immunoelectron microscopic study on the distribution of a cell surface-associated adhesive glycoprotein synthesized by rat ascites hepatoma cells
Authors: Hattori Ryuichi , Ishimaru Yasuji , Kurano Ryoichi , Hayashi Hideo
Published: April 2021

As previously described, a cell surface-associated adhesive factor (AF) was separated from differentiated rat ascites hepatoma AH136B cells (forming cell islands in vivo) and highly purified by chromatography. AF synthesis by the cells was investigated by means of ultrastructural immunoperoxidase cytochemistry. Its synthesis and localization in vivo were observed in the perinuclear spaces, the rough endoplasmic reticulum, the Golgi apparatus, the smooth-membranous vesicles, and the contact region of basolateral cell surfaces of the cells in the islands, and also in the intercellular spaces. No AF synthesis was detectable in the nucleus, the free ribosomes, the mitochondria or the apical non-contacting cell surfaces.

Similar features of AF synthesis and localization were also induced by dissociated AH136B cells in vitro. Upon adhesion of such recovered cells, AF was also localized at the contact surface of the adjacent cells, but not at the non-contacting free surface.
Incomplete contact site a glycoprotein in hl220, a modb mutant of Dictyostelium discoideum
Authors: Gerisch G. , Weinhart U. , Bertholdt G. , Claviez M. , Stadler J.
Published: April 2021

HL220, a modB mutant that lacks a modification of certain membrane proteins of Dictyostelium discoideum, has been shown to aggregate and to form EDTA-stable intercellular contacts typical of aggregating wild-type cells. A developmentally regulated glycoprotein of 80×103 apparent molecular weight has been identified as a target site of adhesion-blocking Fab and thought to be involved in EDTA-stable cell contact formation (Müller & Gerisch, 1978). In the HL220 mutant this glycoprotein is no longer recognized by a modB-specific antibody. Therefore, it has been suggested that the 80×103Mr glycoprotein, or a modification on it, is not required for the EDTA-stable cell contact of aggregating cells.

We show that HL220 synthesizes an equivalent of the 80x lfPMr glycoprotein with an apparent molecular weight of 68×103. The mutant product reacted with certain monoclonal antibodies highly specific for the 80×103Mr glycoprotein in the wild type, and was developmentally regulated like the 80×103MT glycoprotein. These results indicate that the 68×103Mr protein of the mutant lacks a modification, most likely an oligosaccharide residue, the absence of which causes the substantial shift of the apparent molecular weight from 80×103 to 68×103. Monoclonal antibodies that did not react with proteins of the mutant could be classified according to their reactions with different sub-sets of wild-type proteins. These results indicate that the proteins that reacted with either one or the other antibody were not modified by a uniform structure. The modification rather varies from one sub-set of cross-reacting proteins to another,-suggesting differences between the glycosyl residues of the partially cross-reacting proteins.

HL220 cells showed strongly reduced EDTA-stable’contact formation under our conditions. EDTA-sensitive intercellular adhesion was undetectable in the mutant, whereas adhesion of the cells to the substratum appeared to be strengthened.The rear ends of the cells, in particular, were tightly attached to glass or Teflon surfaces.

The mutant cells were capable of responding chemotactically. Propagated excitation waves like those known to be based on periodic cyclic AMP production and relay were clearly seen. Extracellular phosphodiesterase induction by cyclic AMP and phosphodiesterase inhibitor production were normal. These results indicate that the generation of chemotactic signals and the cellular responses to cyclic AMP are not severely affected by the mutation.
Enzymic ribonucleoside reduction at the non-phosphorylated level in Acetabularia
Authors: Groot Egon J. De , Schweiger Hans-Georg
Published: April 2021

The occurrence of an enzyme that catalyses the conversion of cytidine into deoxycytidine was demonstrated in homogenates of Acetabularia. Cytidine was identified as the substrate by comparing cytidine, cytidine 5’-monophosphate, cytidine S’-diphosphate and cytidine 5’-triphos-phate as potential substrates. Experiments with ATP analogues whose inhibitory effect on kinase reactions is well established, supplied evidence that the nucleoside is reduced without a phosphorylation step before the reduction. Further evidence in this line came from incubations with cytidine in the presence of phosphatase and from trap-type experiments in which the effects of excess non-labelled cytidine 5’-phosphate and deoxycytidine, respectively, on the formation of deoxycytidine phosphates from cytidine were studied.
A mutant of Chlamydomonas reinhardtii that lacks the flagellar outer dynein arm but can swim
Authors: Kamiya Ritsu , Okamoto Mitsumasa
Published: April 2021

A new type of Chlamydomonas mutant, which lacks the outer dynein arm but can swim, was isolated. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis showed that four of the ten high-molecular-weight bands of dynein present in the wild-type axoneme are missing or diminished in the mutant axoneme. The mutant has a swimming rate of about 35 μm/s and a flagellar beat frequency of about 25 Hz, both of which are about 1 /2·5 to 1 /3 of those of the wild type. The mutant flagella beat with an asymmetric, cilia-type pattern, similar to the forward-swimming mode of the flagellar beating pattern of the wild type. However, unlike wild-type flagella, the mutant flagella never beat with a symmetrical waveform: when the cells were stimulated by intense light, the mutant transiently stopped beating its flagella, whereas the wild-type cell transiently swam backwards with the two flagella beating with a symmetrical waveform.

Both wild-type and mutant cells could be demembranated by Nonidet P40 and their swimming reactivated by addition of Mg-ATP in the virtual absence of Ca2+. Double reciprocal plots of the beat frequency against ATP concentrations showed a linear relationship for both strains, yielding maximal frequencies of 44 Hz (wild type) and 23 Hz (mutant). The mutant axonemes can be reactivated only when the Ca2+ concentration is lower than 10-6 M: at pCa 4, the wild-type axonemes beat with a symmetrical waveform, but the mutant axonemes showed no movement. These findings indicate that the outer dynein arm is dispensable for flagellar beating of the asymmetric waveform (forward-swimming mode), but not for beating of the symmetrical waveform (backward-swimming mode), and thus suggest the importance of the outer dynein arm in the switching of flagellar waveforms.
Interference reflection microscopy in cell biology: methodology and applications
Authors: Verschueren H.
Published: April 2021

Since its introduction into cell biology by Curtis in 1964, interference reflection microscopy (IRM) has been used by an increasing number of researchers to study cell-substrate interactions in living cells in culture. With the use of antiflex objectives, high-contrast IRM images can now be readily obtained. From the different theories on image formation in IRM that have been put forward, it can be seen that a zero-order interference pattern is generated at high illuminating numerical aperture. This yields information on the closeness of contact between cell and substrate, with only minor perturbation by reflections from the dorsal cell surface. Therefore, the proper use of illuminating apertures is crucial. Nevertheless, IRM images have to be interpreted with caution, especially under thin cytoplasmic sheets. Quantitative IRM is possible only with a mathematical model for finite illuminating aperture interferometry and with an independent measurement of cell thickness for values up to 1 micron. IRM has been applied qualitatively to a large number of cell types, and it seems that there are two universal types of adhesion. Focal contacts are small regions of closest cell-substrate apposition, possibly of immediate contact, that are associated with the distal end of actin filament bundles. They are firm attachment structures that hold the cell in place and in its spread shape. Close contacts are broad areas of reduced cell-to-substrate distance. They are weaker but highly dynamic adhesions that sustain rapid movements of cells or cell parts over the substrate. Although a number of independent observations suggest that adhesion patterns of malignantly transformed cells differ from those of their normal counterparts, there is no simple correlation between malignancy in vivo and altered contact formation in vitro. The adhesion pattern seems to be determined by the locomotory state of the cells rather than by their tissue of origin. Finally, IRM can also be used to enhance contrast in images of fixed preparations.
Growth in cell length in the fission yeast Schizosaccharomyces pombe
Authors: Mitchison J. M. , Nurse P.
Published: April 2021

The cylindrical cells of Schizosaccharomyces pombe grow in length by extension at the ends and not the middle. At the beginning of the cell cycle, growth is restricted to the ‘old end’, which existed in the prev10us cycle. Later on, the ‘new end’, formed from the septum, starts to grow at a point in the cycle that we have called NETO (‘new end take-off’). Fluorescence microscopy on cells stained with Calcofluor has been used to study NETO in size mutants, in blocked cdc mutants and with different growth temperatures and media. In wild-type cells (strain 972) NETO happens at 0·34 of the cycle with a cell length of 9·5μm. With size mutants that are smaller at division, NETO takes place at the same size (9·0–9·5 μm) but this is not achieved until later in the cycle. Another control operates in larger size mutants since NETO occurs at the same stage of the cycle (about 0·32) as in wild type but at a larger cell size. This control is probably a requirement to have completed an event in early G2, since most cdc mutant cells blocked before this point in the cycle do not show NETO whereas most of those blocked in late G2 do show it. We conclude that NETO only happens if: (1) the cell length is greater than a critical value of 9·0–9·5μm; and (2) the cell has traversed the first 0·3–0·35 of the cycle and passed early G2.

NETO is delayed in poor media, in which cell size is also reduced. Temperature has little effect on NETO under steady-state conditions, but there is a transient delay for some hours after a temperature shift. NETO is later in another wild-type strain, 132.

Time-lapse photomicrography was used to follow the rates of length growth in single cells. Wildtype cells showed two linear segments during the first 75% of the cycle. There was a rate-change point (RCP), coincident with NETO, where the rate of total length extension increased by 35%. This increase was not due simply to the start of new-end growth, since old-end growth slowed down in some cells at the RCP. Cdc11.123 is a mutant in which septation and division is blocked at 35 °C but nuclear division continues. The constant-length stage, which occupies the last 25% of the cycle in wild-type cells, also occurs in this mutant at 35°C, so end growth stops and restarts even though there is no septation. In cdc2.33, a mitotic mutant, there is a linear pattern with an RCP in cells held at 35 °C. The RCP is therefore a per10dic event that can take place even though the DNA division cycle is blocked.
Pollen-wall proteins: Electron-microscopiclocalization of acid phosphatase in the intine of Crocus vernus
Authors: Knox R.B. , Heslop-Harrison J.
Published: April 2021

Acid phosphatase has been localized in the wall of the pollen grain of Crocus vernus Wulf at the electron-microscope level by a method using 2-naphthyl thiol phosphate as substrate in a simultaneous coupling reaction with fast blue BBN at pH 50, the product being given electron opacity by osmication.

Activity was found to be concentrated mainly in the intine, and to be associated with ribbon-like or filamentous inclusions believed to be proteinaceous on the basis of other criteria. Some activity was also detectable in the interstices of the exine. The observations confirm the general interpretation of the distribution of wall-held enzyme based upon light-microscopic cytochemistry, and provide the resolution necessary to establish unambiguously that they are associated with protein layers inserted during intine growth.
Mitosis in the fission yeast Schizosaccharomyces pombe as revealed by freeze-substitution electron microscopy
Authors: Tanaka Kenji , Kanbe Toshio
Published: April 2021

Nuclear division in Schizosaccharomyces pombe has been studied in transmission electron micrographs of sections of cells fixed by a method of freeze-substitution. We have found cytoplasmic microtubules in the vicinity of the spindle pole bodies and two kinds of microtubules, short discontinuous ones and long, parallel ones in the intranuclear mitotic spindle. For most of the time taken by nuclear division the spindle pole bodies face each other squarely across the nuclear space but early in mitosis they briefly appear twisted out of alignment with each other, thereby imparting a sigmoidal shape to the bundle of spindle microtubules extending between them. This configuration is interpreted as indicating active participation of the spindle in the initial elongation of the dividing nucleus. It is proposed that mitosis is accompanied by the shortening of chromosomal microtubules simultaneously with the elongation of the central pole-to-pole bundle of microtubules of the intranuclear spindle. Daughter nuclei are separated by the sliding apart of interdigitating microtubules of the spindle at telophase. Some of the latter bear dense knobs at their ends.
Monensin and tunicamycin-induced inhibition of HT29 cell spreading and growth
Authors: El-Battari A. , Muller J. M. , Fantini J. , Bellot F. , Tirard A. , Ducret F. , Marvaldi J.
Published: April 2021

HT29 cells originating from a human colon adenocarcinoma, spread very rapidly after seeding on their own extracellular matrix (ECM). Preincubation of cells with the inhibitor of protein glycosylation tunicamycin (TM) or with the ionophore monensin substantially suppressed cell spreading in serum-free medium without affecting cell adhesion to ECM. Addition of the drugs after cell attachment and spreading inhibited cell growth. TM-treated cells remained viable after 6 days of exposure to 2μgml−1 ′ of TM and resumed their normal growth rate and shape after removing the drug from the medium. On the contrary, monensin inhibition of cell growth was not reversible: after 3 days, cells detached from the ECM and were unable to exclude Trypan Blue. At the ultrastructural level, a swollen Golgi apparatus with numerous vacuoles was observed after treatment for 2h in either TM or monensin-preincubated cells. These results suggest that TM and monensin interfere with the insertion and, or, function of one or more cell surface glycoproteins, possibly interacting with cytoskeleton and involved in cell spreading and growth.
Calcium-transport function of the chick embryonic chorioallantoic membrane : I. in vivo and in vitro characterization
Authors: Tuan Rocky S. , Carson Monica J. , Jozefiak Judith A. , Knowles Kathy A. , Shotwell Barbara A.
Published: April 2021

During chick embryonic development, the chorioallantoic membrane (CAM) is responsible for the mobilization of shell calcium into the embryonic circulation. The calcium-transport function of the CAM was studied here by measuring CAM calcium uptake in vivo and in vitro. The in vivo technique involved the use of an uptake chamber constructed on top of the CAM in situ. The in vitro methods included two systems: CAM tissue disks and cell-free microsomal membranes isolated from the CAM. Analyses using these three assays show that calcium uptake by the CAM exhibited characteristics indicative of active transport, such as temperature dependence, satura-bility, energetic requirement and ion specificity. The data also show that calcium-uptake activities of the CAM increase as a function of embryonic age in a manner coincident with the increased accumulation of calcium by the developing embryo in ovo.
Calcium-transport function of the chick embryonic chorioallantoic membrane ii. functional involvement of calcium-binding protein, Ca2+ -atpase and carbonic anhydrase
Authors: Tuan Rocky S. , Carson Monica J. , Jozefiak Judith A. , Knowles Kathy A. , Shotwell Barbara A.
Published: April 2021

This study aimed to investigate the mechanism of active calcium transport in the chick embryonic chorioallantoic membrane (CAM) by assessing the functional involvement of three previously identified, putative components of the transport pathway. These components are a calcium-binding protein (CaBP), Ca2+-activated ATPase and carbonic anhydrase. Using specific reagents, including antibodies and enzyme inhibitors in vivo and in vitro in CAM calcium uptake assays, it was shown that these biochemically identified components were all functionally involved. The results of these studies also indicate that active calcium uptake by the CAM requires the presence of the CaBP on the cell surface in a laterally mobile manner, while carbonic anhydrase appeared to be a cytosolic component. We further analysed the subcellular location of the calcium-uptake activity by gel filtration and density-gradient fractionation of cell-free microsomes of the CAM and the results suggest that this activity is associated with the plasma membrane.
Spontaneous and induced dome formation by two clonal cell populations derived from a human adenocarcinoma cell line, ht29
Authors: Fantini J. , Abadie B. , Tirard A. , Remy L. , Ripert J. P. , Battari A. El , Marvaldi J.
Published: April 2021

The replacement of glucose by galactose in the culture medium resulted in partial structural and functional enterocytic differentiation of HT29 cells. In order to characterize populations of homogeneously differentiated HT29 cells we have selected two clonal cell lines HT29-D4 and HT29-D9 with the following functional and structural characteristics when grown in a galactose-containing medium: (1) the two clonal cell populations were permanently morphologically differentiated as shown by the presence of mature junctional complexes and a well-organized brush border (especially for HT29-D4 cells); (2) HT29-D4 and HT29-D9 cells were able to form domes early in confluency, which indicated a functional state of differentiation; (3) the process of differentiation was fully reversible when glucose was added to the culture medium.

The induction of domes was investigated in these two cell populations and we demonstrated for the first time that proteolytic enzymes are potent inducers of dome formation. The architecture of domes either obtained spontaneously or induced by proteolytic enzymes was not maintained in the presence of ouabain (a specific inhibitor of the Na+/K+-ATPase).

In conclusion, HT29-D4 and HT29-D9 cells can be maintained permanently in a differentiated state in a glucose-free medium and were able to form domes at confluency. The observation that proteolytic enzymes were able to induce dome formation can help in the comprehension of the mechanism involved in the establishment of the differentiated state.
Alignment of fibroblasts on grooved surfaces described by a simple geometric transformation
Authors: Dunn G. A. , Brown A. F.
Published: April 2021

The response of chick heart fibroblasts to grooved substrata was studied using microfabricated grooves and new measures of shape and alignment derived from the moments of cell shapes. Cell shape and alignment were measured on 23 different sets of regular, parallel grooves, which ranged in width from 1·65 to 8·96μm, and in repeat spacing from 3·0 to 32·0 μm. The grooves were of constant depth, 0·69 μ. Digitized video images were analysed to extract the zero-, first- and second-order moments of the cell shapes, from which were calculated three measures of cell shape, and three measures of cell alignment.

Regression analyses of the measures against parameters of the substratum such as groove width, repeat spacing and the ridge width between grooves show that ridge width is the main parameter affecting cell alignment (alignment being inversely proportional to ridge width), although groove width has a small additional effect. All the differences in cell shape between the different grooves can be summarized to a very good approximation as simple geometrical stretch transformations of the shapes of cells on planar surfaces. Our principal measure of cell alignment, paraxial elongation, is a measure of the necessary transformation. This finding has the interesting biological implication that the shape and orientation adopted by cells, in response to the grooves, are not governed by independent cellular mechanisms.
Intermediate filament proteins in classic and variant types of small cell lung carcinoma cell lines: A biochemical and immunochemical analysis using a panel of monoclonal and polyclonal antibodies
Authors: Broers J. L. V. , Carney D. N. , Rot M. Klein , Schaart G. , Lane E. B. , Vooijs G. P. , Ramaekers F. C. S.
Published: April 2021

The intermediate filament protein (IFP) characteristics of a panel of lung cancer cell lines including adenocarcinoma (two cell lines) and small cell lung cancer (SCLC, three classic and three variant cell lines) were examined using one- and two-dimensional gel electrophoretic techniques, immunocytochemical techniques and immunoblotting assays. A panel of 28 monoclonal and polyclonal antibodies to the five different types of IFP were used. The results of our studies indicate that these human lung adenocarcinoma, classic SCLC and variant SCLC cell lines can be differentiated on the basis of their pattern of IFP.

The main conclusions from this study can be summarized as follows. (1) The two adenocarcinoma cell lines contain cytokeratins 7, 8, 18, and sometimes 19, next to vimentin intermediate filament (IF). (2) The three classic-type SCLC cell lines contain only cytokeratin IFs but not vimentin IF or neurofilaments (NFs). Cytokeratin polypeptides 7, 8, 18 and 19 could be detected. (3) All three variant-type SCLC cell lines do not contain detectable amounts of cytokeratins. In contrast, two out of three variant SCLC cell lines contain neurofilament proteins. All three varianttype SCLC cell lines contain vimentin IF. (4) Using immunoblotting assays with monoclonal and polyclonal antibodies to defined NF proteins the presence of the 68×103Mr and the 160×103Mr NF polypeptide could be demonstrated in two variant SCLC cell lines.

As patients with SCLC-variant phenotype have a poorer prognosis after cytotoxic therapy than patients with‘pure’ SCLC, the use of antibodies to IFP in staining fresh lung tumours, especially anaplastic ones, may differentiate the two subtypes of SCLC. Such a distinction would have a major impact on therapy selections and may be of prognostic importance.
Development of a three-dimensional extracellular matrix synthesized by human diploid fibroblasts in vitro
Authors: Qwarnström Eva E. , Page Roy C.
Published: April 2021

Development and maturation of an extracellular matrix, synthesized by human gingival fibroblasts, have been studied microscopically. Pairs of demineralized, fibronectin-coated slices of human tooth root, 300 μm thick, were placed on confluent cell layers, defining a 0·5 mm wide space. The cultures were grown under standard conditions with ascorbic acid (50μgml−1) added daily. At various times up to 13 weeks, the cultures were fixed and the samples prepared for light and electron microscopy.

Cells from the monolayer became attached to, and migrated up, the vertical root surface and, during the time studied, completely filled the space between the root slices with an extracellular matrix. A close association was seen between the cell membrane and collagen fibres in the demineralized surface initially. A thin layer of fibrillar material was deposited between the cell and the vertical surface, and eventually an extracellular matrix surrounding the cells and attaching to the root surface was present. Samples fixed in the presence of Ruthenium Red showed intense staining of the fibrillar material, indicating the presence of anionic molecules. Additional cells migrated onto the newly synthesized matrix and up the root surface. Growth of the fibrillar networks on either side, horizontally and vertically, continued and, eventually, an extracellular matrix attaching to the vertical surfaces completely filled the previously empty space. Immunocytochemical staining showed that the matrix contained hyaluronic acid, chondroitin sulphate, dermatan sulphate and fibronectin at this time. Collagen fibres were observed at 6 weeks, and at later times collagen types I, III and V were the primary matrix components.

The fibroblasts attaching to the root slice and those present at the edge of the matrix had an elongated, polar form. The cells within the matrix frequently showed a stellate appearance with numerous extended processes, in contact with fibrillar material or collagen fibres. Fibroblast processes were at later times seen to enclose bundles of collagen fibres and to mediate cell-to-cell contact, occasionally via desmosome-like structures.

The structure and composition of the matrix and the appearance and apparent behaviour of the cells were similar to that observed in the healing wound. This system thus could provide a model for studying various aspects of regeneration of extracellular matrix.
Intra- and extracellular distribution of an endogenous lectin during erythropoiesis
Authors: Harrison F. Lynne , Catt James W.
Published: April 2021

An endogenous β-galactoside-specific lectin has previously been isolated from rabbit bone marrow. The quantification of extracted lectin now indicates that approximately 75% of the lectin is intracellular in marrow. Indirect immunofluorescence studies show the extracellular lectin is associated with the erythroblast cell surface and is also found in some acellular areas of the marrow stroma. At enucleation, lectin surrounds the extruded nucleus while some residual lectin is observed in the cytoplasm of circulating reticulocytes and erythrocytes.
Structurally related Bacillus thuringiensis δ-endotoxins display major differences in insecticidal activity in vivo and in vitro
Authors: Knowles Barbara H. , Francis Philippa H. , Ellar David J.
Published: April 2021

Many strains within the 22 serotypes of Bacillus thuringiensis produce crystal δ-endotoxins with slight differences in their insecticidal toxicity spectrum in vivo. Since the basis of this specificity is unknown, we chose to compare the activity of δ-endotoxins from three strains: B. thuringiensis var. kurstaki HD-1, var. aizawai HD-249 and var. thuringiensis HD-350, both in vivo and on insect cell lines in vitro. Immunoblotting with antisera to activated var. kurstaki Pl lepidopteran toxin revealed antigenic cross-reaction with the 130×103Mr toxin of var. aizawai, and with polypeptides of 130 and 138(×103)Mr from var. thuringiensis. In addition, crystals from var. kurstaki and var. aizawai contained an antigenically related 63xlO3Afr protein that did not cross-react with antisera to the 130× 103Mr component.

Bioassays on Pieris brassicae larvae (Lepidoptera) and Aedes aegypti larvae (Diptera) indicated that the 130×103Mr protein of var. kurstaki, and the 138 plus 130(×103)Mr components of var. thuringiensis killed only P. brassicae, while the 130×103.Mr protein of var. aizawai and the 63×103Mr proteins of var. aizawai and var. kurstaki were toxic to both P. brassicae and A. aegypti.

Activation of the 130 and 138 (×103)Mr proteins of the three varieties of B. thuringiensis with insect gut proteases yielded active products of 50— 60 (× 103)Mr. Assay of these products on a range of lepidopteran and dipteran cell lines revealed very different toxicity spectra: var. kurstaki killed only one lepidopteran line, var. thuringiensis killed two lepidopteran lines, while var. aizawai was cytolytic to all of the lepidopteran and most of the dipteran cell lines tested, reflecting its broader spectrum in vivo.

Thus we have shown that antigenic cross-reaction of B. thuringiensis δ-endotoxins does not necessarily imply a similar toxicity spectrum in vivo or in vitro.
Microinjection of immaturin rejuvenates sexual activity of old Paramecium
Authors: Haga Nobuyuki , Karino Setsuko
Published: April 2021

The sexual activity of aged Paramecium that underwent about 700 fissions after conjugation was remarkably decreased. Microinjection of a soluble cytoplasmic fraction prepared from young immature cells (about 20 fissions after conjugation) restored the sexual activity of the aged cells to that of presenescent mature cells. The factor responsible for the recovery of the sexual activity’ was shown to have the same characteristics as immaturin in terms of molecular size and net charge. Rejuvenation of sexual activity lasted up to 20 fissions after microinjection. However, cell division rate or fertility after self-conjugation was not increased by the microinjection. Our finding indicates that the decrease in sexual activity caused by aging was not due to defects in the genes for mating substances but to a decrease in the activity of a soluble protein responsible for the expression of mating activity. This is the first description of the restoration of a senescent defect by microinjection of young cytoplasmic components into Paramecium.
The mobility of intramembrane particles in non-haemolysed human erythrocytes: Factors affecting acridine-orange-induced particle aggregation
Authors: Lelkes Gábor , Fodor Ildikó , Lelkes György , Hollán Susan R.
Published: April 2021

It has previously been shown that reversible intramembrane particle aggregation can be induced in non-haemolysed human erythrocytes. This phenomenon, which can be induced by the cationic dye Acridine Orange, has been further investigated using different experimental conditions that are expected to influence the rate of aggregation of the particles.

In addition to the concentration of the dye, the rate of aggregation was also found to be dependent on the extracellular and intracellular pH, as well as on the type of buffer used. While lowering the pH of the Acridine Orange solutions resulted in decreased particle clustering, low intracellular pH increased and elevated intracellular pH decreased particle aggregation. Furthermore, at a given dye concentration and a given pH, Acridine Orange caused more intense aggregation in Tris-buffered saline than in isotonic phosphate buffer or phosphate-buffered saline. Under appropriate conditions Acridine Orange caused significant particle aggregation at concentrations as low as 0·25 mH within 30 s. During this period only discocyte-stomatocyte transformation occurred; neither agglutination nor vésiculation of the erythrocytes could be detected.

Treatment of the erythrocytes with Diamide (Serva), which cross-links spectrin via disulphide bridges and thereby reduces lateral diffusion of integral membrane proteins over large distances, had no inhibitory effect on Acridine-Orange-induced particle aggregation. Heating the erythrocytes to 50 °C, at which temperature denaturation of spectrin and fragmentation of the erythrocytes occur, and subsequently incubating them in Acridine Orange at room temperature, caused an almost maximal rate of particle aggregation within 10—30 s, without haemolysis.

The possible mechanism and significance of the particle aggregation phenomenon are discussed.
Substrate hydroxylation and cell adhesion
Authors: Curtis A. S. G. , Forrester J. V. , Clark P.
Published: April 2021

Adhesion of BHK cells to a variety of polymer surfaces carrying measured densities of hydroxyl and carboxyl groups was studied. The effects on cell adhesion of blocking hydroxyl groups by acetylation and carboxyl groups with diazomethane were measured. Hydroxyl groups were required for cell adhesion, though the very high surface densities of these groups diminished cell adhesion. The optimal surface density of OH groups for BHK adhesion was 2000 per 1× 10−11 cm2. Carboxyl groups slightly inhibit cell adhesion, since blocking of these groups by methylation increased adhesion. The role of oxidizing systems of cellular origin in conditioning of the substrate, in serum-free conditions, was demonstrated for leucocytes and BHK cells, in particular by the result that oxidizable substrates such as phytane and poly(l,2-butadiene) could be made suitable for cell adhesion by contact with cells.
Spreading behaviour of cultured fibroblasts from carriers of duchenne muscular dystrophy
Authors: Pizzey John A. , Witkowski Jan A. , Jones Gareth E.
Published: April 2021

Cultured skin fibroblasts from patients with Duchenne muscular dystrophy (DMD) are more sensitive than normal cells to prolonged exposure to the ionophore monensin. In a cell spreading assay in which cells were preincubated with monensin and subsequently allowed to adhere to and spread on a glass substratum in serum-free medium for 100 min, the mean transformed cell area of normal and DMD cells was 5 ·97 ±0 ·11 and 5 ·29 ± 0 ·03, respectively. Cultured fibroblasts from carriers of DMD yielded a value of 5 ·59 ± 0 ·03, which is intermediate between, and significantly different from, the values for both normal and DMD cultures. This result would be predicted on the basis of random X-chromosome inactivation in female carriers of this disorder. However, comparison of DMD carrier cell spreading data with data obtained from pooled and summated measurements taken from separate experiments using either normal or DMD fibroblasts suggest a more complex situation. Examination of the variance of the means of cell area for the true carrier population and the summated normal and DMD population provides evidence suggesting that some form of cellular interaction may occur between the two cell genotypes in culture.
Temperature and ph govern the self-assembly of microtubules from unfertilized sea-urchin egg extracts
Authors: Suprenant Kathy A. , Marsh John C.
Published: April 2021

A new method for microtubule purification from unfertilized sea-urchin eggs was developed in order to obtain large quantities of calcium- and cold-labile microtubules that contained microtubule-associated components important for mitosis. By taking into consideration the pH, ionic composition of egg cytoplasm, and the physiological temperature for growth of the Pacific coast sea-urchin Strongylocentrotus purpuratus, methods were developed for the assembly of intact microtubules directly from unfertilized egg extracts. The microtubules obtained by cycles of temperature-dependent assembly and disassembly are composed of tubulin and abundant microtubule-associated proteins. These microtubules are cold- and calcium-labile and assemble at a critical protein concentration of 0 ·11 mg ml−1 at 24°C. The yield of microtubule protein obtained by this new method is equivalent to that obtained with taxol (6—8mg/20ml packed eggs). Microtubules that have been fixed and prepared for electron microscopy are decorated with large, globular projections that are attached to the microtubule by thin stalks.
Monensin inhibits secretion of extracellular matrix and the spreading of mesoderm cells in the early chick embryo
Authors: Sanders E. J. , Chokka P.
Published: April 2021

Chick embryos in culture were treated with the secretion-inhibiting ionophore monensin at the gastrulation stage of development. After treatment for 5h at a concentration of 10−5M the embryos showed a drastic reduction in the tissue space between the upper and lower epithelia, and reduced spreading of the mesoderm cells that occupied this space. The basement membrane, to which many mesoderm cells are normally attached, showed varying degrees of disruption, which permitted blebbing of the overlying epithelium. Intracellularly, the treatment caused extensive vacuolization in all tissues. These results were consistent with the effects expected by removal of hyaluronic acid from the extracellular space, and this was confirmed by demonstrating a sharp reduction in glucosamine incorporation in this region. Examination of the effects of monensin on isolated mesoderm cells in culture using interference reflection microscopy indicated that the spreading of these cells was reduced independently of the changes in the extracellular matrix of the embryo. That this was probably due to the inhibition of cell surface fibronectin secretion was shown by demonstrating severe changes in the distribution of this glycoprotein using the immunofluorescent technique. It is concluded that the effects of monensin on this intact, developing system are due primarily to the disruption of hyaluronic acid secretion, but that disrupted fibronectin synthesis contributes to the reduced spreading of mesoderm cells.
Cell activation and adhesion
Authors: Curtis Adam
Published: April 2021

It has long been known that the adhesiveness of leucocytes and blood platelets can be changed from a state of non-adhesion to very strong adhesion to each other, and to many surfaces, in a very short time. The agents that produce such changes are prostaglandins, thromboxanes and leukotrienes (see for example, Hoover et al. 1984), agonists affecting production of these substances and also those that affect the diacyl-glycerol-inositol triphosphate pathways (see Nishizuka, 1984). Evidence has recently emerged (Dunham et al. 1983; Dunham & Weissman, 1986) showing that sponge cells can have their adhesion activated by agents that affect calcium relationships of cells through diacylglycérol—ITP pathways. Brief pulses of low levels of calcium ions activate adhesion of these cells, phorbol myristoyl acetate and a calcium ionophore act synergistically in aiding cell adhesion. The activating agents can be effective at nanomolar concentrations. It is of interest to ask whether similar activation phenomena are more widespread than suspected.
Schizosaccharomyces Pombe mutants affected in their division response to starvation
Authors: Young Paul G. , Fantes Peter A.
Published: April 2021

Schizosaccharomyces pombe mutants have been selected on the basis of an altered response to nutritional stimulation of cell division (changed division response, cdr). Two new loci (cdr1 and cdr2) were identified and characterized. When suspended in nitrogen-free medium wild-type cells underwent stimulated rates of division and became reduced to approximately 30 % in protein content with a concomitant 3·6-fold increase in cell number after 24 h starvation, cdr cells had significantly smaller increases in cell number. The ratio of starved/unstarved protein content was higher for the cdr strains than for the wild type, cdr cells were also affected in their response to nitrogen-source shifts from proline to glutamate (or vice versa) or when shifted from serine phosphate to inorganic phosphate, showing that the alteration in division response was not restricted to nitrogen metabolism. Upon nitrogen starvation wild-type cells arrested prior to the cdc10 execution point, whereas cdr cells arrested later in the cell cycle. cdc25–22 cdr1 or cdr2 double mutants grew very slowly and were extremely elongated at all temperatures; the restrictive temperature was reduced to 27°C. wee1 was epistatic to cdr mutations with respect to cell length at the cell plate stage. cdr+ genes are postulated to play a role in the nutritional modulation of the mitotic size control.
Expression of an endogenous galactose-binding lectin in the early chick embryo
Authors: Zalik Sara E. , Thomson Lawrence W. , Ledsham Irene M.
Published: April 2021

The gastrulating chick blastoderm contains lectin activity specific for β-D-galactoside groups. The galactose-binding lectin isolated by affinity chromatography on p-aminophenyl-β-D-lactoside separates into two bands when studied by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. One of these Ln has a relative molecular mass of 70(±2)×103 while the other L1 is a polypeptide that migrates with the dye front in 10% gels. We have prepared an antiserum against this lectin preparation and have affinity-purified antibodies against L1. When embryos at stages 3—7 were examined by immunofluorescence using the affinity-purified antibodies, lectin was expressed in cells at the lowest portions of the primitive streak as well as in cells migrating laterally from this region to form the endoderm. Lectin was also expressed by the cells of the extra-embryonic endoderm and the primordial germ cells of the proximal area opaca. In transfers of gradient gels stained with affinity-purified antibodies against LIthis lectin had an approximate molecular weight of 6·5 ×103. Our results indicate that this lectin is expressed in areas that are undergoing cell spreading.
Immunogold localization of placental lactogen and the sbu-3 antigen by cryoultramicrotomy at implantation in the sheep
Authors: Morgan G. , Wooding F. B. P. , Brandon M. R.
Published: April 2021

In the sheep, granulated trophectodermal binucleate cells (BNC) appear at implantation 16 days post coitum (dpc) and persist throughout pregnancy. Conventional immunocytochemical techniques at both light and electron microscope levels have indicated the presence of the ovine placental lactogen (oPL) hormone in the granules but no earlier than 22 dpc, when the level was very low. Immunofluorescent studies using glycolmethacrylate sections between 15 and 55 dpc suggest a completely different distribution of oPL restricted to uninucleate cells with none in the BNC. Using the most sensitive method available, immunocytochemistry on ultrathin frozen sections, the results in this paper demonstrate that BNC granules contain oPL at their earliest appearance (16–17 dpc). No significant localization was found in any uninucleate cell. In contrast, another molecule, the SBU-3 antigen, which is demonstrated in BNC granules later in pregnancy, is not present at the earliest stages but appears between 24 and 28 dpc coincident with the development of the foetal cotyledonary villi. The significance of these results for BNC function are discussed briefly.