bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence that tubulin forms an integral membrane skeleton in molluscan gill cilia
Authors: Stephens R. E. , Oleszko-Szuts S. , Good M. J.
Published: April 2021

Controlled extraction of intact gill tissue, isolated cilia or reconstituted membrane vesicles with Nonidet P-40 at >4 times the critical micelle concentration, or with octyl glucoside at the critical micelle concentration, delipidates the membrane, leaving a membrane remnant or skeleton of membrane tubulin and associated proteins. This skeleton consists of a disordered reticular protein network in reconstituted membrane vesicles and a similar but more compact sleeve in cilia of extracted tissue. The membrane skeleton is closely apposed to the axoneme and is attached to the outer doublets by fine radial bridges having a 20–24 nm longitudinal periodicity, supporting earlier observations made utilizing a lipophilic cross-linking agent. Higher concentrations of detergent solubilize the membrane tubulin-protein complex, producing 5–10 nm particulate material of low sedimentation coefficient. Dilution of an octyl glucoside solution to below the critical micelle concentration results in disappearance of the particles and reformation of the membrane, indicating that the particles are protein-detergent micelles and not denatured protein. Freeze-fracture electron microscopy reveals no comparablesized natural particles in the ciliary membrane proper. The reticular material of the membrane skeleton contains tubulin, demonstrated on Low-icryl K4M thin sections by a rabbit polyclonal antibody to sea-urchin egg cytoplasmic tubulin, using gold-labelled secondary antibody. Minimal cross-reactivity is detected prior to Triton-delipi-dation, suggesting that most membrane tubulin antigenic sites are buried within the bilayer and that the tubulin is not simply adsorbed to the lipid bilayer.
Chloral hydrate disrupts mitosis by increasing intracellular free calcium
Authors: Lee Greta M. , Diguiseppi Jim , Gawdi Govind M. , Herman Brian
Published: April 2021

In examining how chloral hydrate affects mitosis, we found that extracellular application of 0·l % chloral hydrate produced an abrupt rise in cytosolic free Ca2+. Digitized fluorescence microscopy of Fura-2-loaded, mitotic and interphase PtK cells revealed that Ca2+ rose 15 s after chloral hydrate application, peaked within 1 min at a concentration two-to sevenfold above the basal level and then slowly dropped. Bathing cells in 0·l% chloral hydrate caused metaphase spindles to shorten, starting in 1–2 min, and inhibited spindle elongation without affecting chromosome-to-pole movement during anaphase, as determined by phase-contrast observation of living cells. Spindle elongation and chromosome movement were unaffected by intracellular injection of 7·5% chloral hydrate. Extensive mitotic microtubule breakdown occurred after cells were bathed for 7 min in 0·l% chloral hydrate, while interphase microtubules were unaffected as determined by immunofluorescence. The chloral hydrate-induced microtubule breakdown and metaphase spindle shortening were prevented by 10mM-CoCl2, which has previously been shown to block Ca2+ influx and to stabilize microtubules in vitro. These results imply that disruption of mitotic spindle function and structure by chloral hydrate is due to a rise in cytosolic free Ca2+, and also indicate that mitotic microtubules are more Ca2+-labile than interphase microtubules.
The translocation of mitochondria along insect ovarian microtubules from isolated nutritive tubes: a simple reactivated model
Authors: Stebbings II. , Hunt Cherryl
Published: April 2021

Nutritive tubes, the microtubule-based translocation channels that link the trophic tissue to the developing oocytes in the ovaries of hemipteran insects, have been isolated and examined using video-enhanced differential interference contrast microscopy. When viewed in this way the nutritive tubes are seen to fray into linear strands, which, on the addition of exogenous ATP, support the translocation of particles along their lengths. The movement is also seen with GTP but not AMP-PNP. It is not affected by the addition of inhibitors of dynein or of energy metabolism. Electron microscopy shows the strands to consist of bundles of parallel microtubules of different sizes and the moving particles to be mitochondria. Comparisons are drawn between the movement of mitochondria along isolated insect ovarian microtubules and the reported translocation of vesicles along microtubules from squid axoplasm. The simplicity of the insect system is emphasized. The fact that it can be isolated easily and characterized biochemically makes it potentially valuable for investigating microtubule-based translocation.
Polo, a mitotic mutant of Drosophila displaying abnormal spindle poles
Authors: Sunkel Claudio E. , Glover David M.
Published: April 2021

Neuroblast cells in larvae homozygous for mutant alleles of the locus polo show a high frequency of metaphases in which the chromosomes have a circular arrangement, and anaphase figures in which chromosomes appear to be randomly oriented with respect to at least one of the spindle poles. These defects appear to lead to the production of polyploid cells. Sex chromosome disjunction is affected in male meiosis, primarily in the second division, and the meiotic spindles of living cells are abnormal. One allele is a larval lethal, whereas another is semi-lethal with about 7% of homozygotes surviving as adults. Embryos from homozygous polo females have aberrant mitotic spindles that are highly branched and have broad poles. Immunofluorescence studies with an antibody that recognizes an antigen associated with the centrosome indicate that the organization of this organelle is disrupted in the mutant embryos.
Transmission of gametic nuclei through a fertilization tube during mating in a primitive dinoflagellate,Prorocentrum micansEhr
Authors: Bhaud Yvonne , Soyer-Gobillard Marie-Odile , Salmon J. M.
Published: April 2021

In the dinoflagellate Prorocentrum micans Ehr., changing the culture medium (switching from Erdschreiber’s to Provasoli’s medium) provokes the appearance of individuals that are morphologically different from the normal vegetative forms. Observations and microspectrofluorimetric measurements made in vivo of the relative amounts of nuclear DNA show that these forms are sexual forms; unlike the situation in dinoflagellate species that are known to be sexual, the male and female gametes of P. micans do not fuse. Cells playing the role of isogametes and containing q DNA pair and form a fertilization tube by means of which a donor cell (♂) injects its nucleus into a recipient cell (♀). After conjugation, the zygote containing 2q DNA replicates and thereafter contains 4q DNA. Two successive meiotic divisions lead to the formation of a tetrad in which each nucleus contains q DNA. Cells released from the tetrad seem to be adapted to Provasoli’s medium and vegetative divisions occur again. The characteristics of sexual reproduction in those dinoflagellates in which this phenomenon has been described are reviewed and discussed.
Centriolar location during blood cell spreading and motion in vitro: An ultrastructural analysis
Authors: Gudima G. O. , Vorobjev I. A. , Chentsov Yu S.
Published: April 2021

Different motile blood cells behave differently on a glass surface. Macrophages go through all the stages of spreading described previously for fibroblasts; granulocytes become polarized after a short stage of radial spreading, while the polarization of lymphocytes takes place immediately upon attachment. The active centrioles of the cells we have studied orient their distal ends towards the upper cell surface. The centrioles in blood cells have a different location with respect to the nucleus and the leading edge of the cell: in macrophages the centrioles are situated mostly anterior to or at the side of the nucleus; in granulocytes they lie between the nuclear segments; and in lymphocytes they are positioned strictly posterior to the nucleus, in the uropod. In each case, however, the centrioles are localized in the central region of the cytoplasm. Their alignment does not appear to be related to the blood cells’ random motion in vitro.
Positive regulation of melanin pigmentation by two key substrates of the melanogenic pathway, l-tyrosine and l-dopa
Authors: Slominski Andrzej , Moellmann Gisela , Kuklinska Elizabeth , Bomirski Andrzej , Pawelek John
Published: April 2021

We describe results demonstrating the positive regulation of melanogenesis by two substrates of the melanogenic pathway. We have found that L-tyrosine and L-dihydroxyphenylalanine (L-dopa), whose metabolic fates are affected by the activity of that pathway, can also act as its regulators. In living pigment cells, tyrosinase (EC 1.14.18.1), a crucial and rate-limiting enzyme of melanogenesis, acts in subcellular organelles known as melanosomes. Melanin is laid down only in these organelles. We demonstrate that supplementing Ham’s F-10 medium with additional L-tyrosine or L-dopa during the culture of amelanotic Bomirski hamster melanoma cells results in a rapid increase in melanin formation, which is not simply due to greater availability of substrate. There is a rapid increase in tyrosinase activity and a large scale synthesis of melanosomes. The effects of L-tyrosine and L-dopa are prevented by the addition of cycloheximide. The actions of L-tyrosine and L-dopa are specific in that under similar conditions D-tyrosine, D-dopa, N-acetyl-L-tyrosine, L-phenylalanine, L-tryptophan and L-valine have little or no effect. The two substrates, L-tyrosine and L-dopa, appear to act through related but distinct mechanisms. Our findings provide an example of a little-known phenomenon: regulation of a differentiated eukaryotic phenotype through positive control by substrates in the pathway.
Identifying characteristics of the haematopoietic precursor cell
Authors: Murphy M. J. , Bertles J. F. , Gordon A. S.
Published: April 2021

Studies are reported utilizing the known ability of homologous marrow cells to prevent death in otherwise lethally X-irradiated mice by ‘colonizing’ their spleens. The proportion of marrow cells functioning as progenitor cells attains a peak value during post-hypoxic polycythaemia. On the third day post-hypoxia, bone marrows of mice were harvested and prepared for density-gradient centrifugation. The relatively pure fractions of marrow cells were concentrated on the basis of their density and directly injected into the surgically exteriorized spleens of mice which had received an otherwise lethal dose of X-irradiation 48 h previously. Animals were sequentially killed from 15 min to 8 days later and spleens were prepared for examination by light and electron microscopy.

Eighty to 95 % of the light-density fraction (p = 1·0200–1·0440) was composed of large mono-nuclear cells characterized by a leptochromatic nucleus with pachychromasia at the nuclear envelope, one or more prominent nucleoli, sparse endoplasmic reticulum, a moderate number of mitochondria, a conspicuous Golgi zone and a centrosome in the region of a nuclear indenta-tion. Cells of this light fraction, devoid of erythroid elements, were competent in colonizing spleens (erythrocyte, granulocyte and megakaryocyte series) in contrast to the cells present in fractions of greater density.

The name ‘haematopoietic precursor cell’ (HPC) is tentatively used in describing this cell which is discussed in the light of some of the proposed models of haematopoiesis.
Cellular surfaces of amphibian atrial muscle
Authors: Lorber V. , Bertaud W. S.
Published: April 2021

The cells of the amphibian atrial myocardium contain many so-called micropinocytosis vesicles, irregularly distributed along the cell membrane. In freeze-etch replicas they appear as pits when the cell membrane is seen as from an external view point, or as irregular stumps, when viewed as from an internal or cytoplasmic aspect of the membrane. In occasional specimens in which the fracture has traversed a sagittal plane the vesicles exhibit the flaskshaped appearance noted in sectioned material. In sections from tissue fixed in aldehydes and post-fixed in an osmium-lanthanum solution, the vesicles contain lanthanum whether or not they appear to be attached to the surface membrane. Since this agent is an extracellular marker, administered after cell death, this observation is taken as an indication that all the vesicles communicate with the extracellular space, and that failure to visualize the connexion is merely a consequence of the plane of sectioning. Vesicles not connected to the membrane, and free of lanthanum could not be found, suggesting that if micropinocytosis does occur, abscission is an infrequent event, and/or the vesicles are very short-lived following separation from the membrane.

The possibility that these organelles may act as an extension of the cell membrane and serve to enlarge its area has been suggested. This implies that calculations of cell membrane area based on the assumption of a smooth surface may be in error, though the magnitude of the error cannot be estimated from the present information.
Effects of hnk-1 monoclonal antibody on the substratum attachment and survival of neural crest and sclerotome cells in culture
Authors: Sanders E. J. , Cheung E.
Published: April 2021

The sclerotome portion of the differentiating embryonic chick somite becomes infiltrated by neural crest cells prior to its dispersal towards the embryonic axis. This means that sclerotome cells explanted into culture for the purpose of examining their interactions in vitro are contaminated with a proportion of neural crest cells. The purpose of this study was to explore the neural crest cell adhesion epitope recognized by the HNK-1 monoclonal antibody and ways in which this antibody can be used to eliminate neural crest cells from mixed culture by selective cytotoxicity. Immunofluorescence technique, under the conditions used here, indicated that the antibody appeared to stain all the mesenchymal (i.e. neural crest) cells emigrating from pieces of embryonic neural tube in culture. Examination of the effects of HNK-1 suggests that the antibody interacts with substratum-binding sites on the neural crest cell surface. On fibronectin-coated substrata the antibody tended to inhibit neurite outgrowth but left the cells relatively well spread, while on laminin substrata the effect was to discourage both neurite extension and cell spreading, causing cell retraction. These results suggest that the cell surface epitope recognized by HNK-1 influences neurite outgrowth, neurite adhesiveness or both. Failure of cell spreading on laminin suggests interaction with the laminin binding sites on the cell body. Elimination of the crest cells from mixed culture with sclerotome was achieved by culturing the cells in the presence of HNK-1 antibody and complement during the period required for complete cell outgrowth from the sclerotome explant. This treatment resulted in cultures that, on further incubation, were entirely HNK-1 negative and that failed to show either the presence of neurite-bearing cells or of cells reacting positively to anti-neurofilament monoclonal antibody. We thus appear to have succeeded in removing the crest cells from the mixed population.
Initiation of the cortical reaction in hamster and sheep oocytes in response to inositol trisphosphate
Authors: Cran D. G. , Moor R. M. , Irvine R. F.
Published: April 2021

Microinjection of inositol 1,4,5–trisphosphate into sheep and hamster oocytes induces secretion of cortical granules in a dose-dependent manner. In the sheep, this effect is strongly pH-dependent with minimal exocytosis taking place at pH 6·8 but a full cortical reaction occurring at pH 8·0. Exocytosis in the hamster is also affected by the pH of the external medium but to a lesser extent. Injection of GTPγS also induces exocytosis in both species but is more effective in the hamster. It is suggested that inositol metabolism stimulated by sperm-egg interaction with a GTP-binding protein may be part of the mechanism leading to cortical granule exocytosis and that this may be modulated by the external pH.
Identification of renin in resident alveolar macrophages and monocytes: HPLC and immunohistochemical study
Authors: Dezsö Balázs , Nielsen Arne Hoj , Poulsen Knud
Published: April 2021

Normal, unstimulated alveolar macrophages (AM) and monocytes (Mo) from both mice and rats have been shown to exhibit an angiotensin-I-forming enzyme. This protease has a similar or identical molecular weight to plasma renin as judged by HPLC analysis. In addition, the enzyme activity could be almost completely inhibited by a specific antibody to pure mouse submaxillary gland renin. This indicates that the angiotensin-I-generating protease in AM and Mo is identical to true renin. These results are in accordance with those of immunocytochemical studies, since the cells sharing immunoreactive renin are macrophages and monocytes. The low percentage of renin positive cells may indicate a functionally different and probably distinct subpopulation of AM and Mo.

The presence of renin and angiotensins and angiotensin-I-converting enzyme in normal macrophages and monocytes seems to be common. Hence, the data presented raise the possibility that an intracellular renin-angiotensin system may infact exist in a certain subpopulation(s) of normal macrophages and monocytes.
Immunochemical and biochemical comparisons between embryonic chick bone marrow and epiphyseal cartilage chondroitin/dermatan sulphate proteoglycans
Authors: Sorrell J. Michael , Mahmoodian Fatemeh , Caterson Bruce
Published: April 2021

Chrondroitin sulphate proteoglycans obtained from embryonic chick bone marrow and epiphyseal cartilage were compared using immunochemical and biochemical analyses. Proteoglycans from each tissue, separated on CsCl density gradients, under dissociative conditions, into high (1·6 gml−1), medium (l·5gml−1) and low (l·4gml−1) buoyant density fractions, were immunochemically analysed, using a panel of monoclonal antibodies that specifically recognize chondroitin 4-/dermatan sulphates, chondroitin 6-sulphate, keratan sulphate, the hyaluronate binding region present on connective tissue proteoglycans, and link protein. The same antibodies were used in Western blot analyses to detect intact proteoglycan monomers and core proteins that had been fractionated by agarose-poly-acrylamide and by sodium dodecyl sulphate-poly-acrylamide gel electrophoresis. Specific differences between marrow and cartilage proteoglycans were detected. In CsCl gradients, marrow proteoglycans displayed a higher degree of heterogeneity in terms of buoyant densities and hexuronate distribution.

Keratan sulphate chains were constituents of the majority of ‘large’ proteoglycans in the marrow; however, a portion of the large proteoglycans in marrow middle buoyant density fraction either lacked keratan sulphate chains or were substituted with a form different from that found on cartilage proteoglycans. Marrow lacked ‘small’ chondroitin/dermatan sulphate proteoglycans that were present in cartilage and contained a more heterogeneous population of proteoglycans, particularly in the lower buoyant density fractions. Both marrow and cartilage were similar in that they contained, as their major components, large, aggregating proteoglycans and link proteins that were immunochemically and biochemically identical. The significance of these differences between marrow and cartilage proteoglycans remains to be determined, but they may, in part, be responsible for imparting unique characteristics to the haematopoietic extracellular matrices.
The cytoskeletal elements of human retinal pigment epithelium: in vitro and in vivo
Authors: Mckechnle Nicol M. , Boulton Mike , Robey Helen L. , Savage Felicity J. , Grierson Ian
Published: April 2021

The cytoskeletal elements of normal (in situ) and cultured human retinal pigment epithelium (RPE) were studied by a variety of immunocytochemical techniques. Primary antibodies to vimentin and cytokeratins were used. Positive immunoreactivity for vimentin was obtained with in situ and cultured material. The pattern of reactivity obtained with antisera and monoclonals to cytokeratins was more complex. Cytokeratin immunoreactivity could be demonstrated in situ and in cultured cells. The pattern of cytokeratin expression was similar to that of simple or glandular epithelia. A monoclonal antibody that specifically recognizes cytokeratin 18 identified a population of cultured RPE cells that had particularly well-defined filamentous networks within their cytoplasm. Freshly isolated RPE was cytokeratin 18 negative by immunofluorescence, but upon culture cytokeratin 18 positive cells were identifiable. Cytokeratin 18 positive cells were identified in all RPE cultures (other than early primaries), regardless of passage number, age or sex of the donor. In post-confluent cultures cytokeratin 18 cells were identified growing over cytokeratin 18 negative cells, suggesting an association of cytokeratin 18 immunoreactivity with cell proliferation. Immunofluorescence studies of retinal scar tissue from two individuals revealed the presence of numerous cytokeratin 18 positive cells. These findings indicate that RPE cells can be identified by their cytokeratin immunoreactivity and that the overt expression of cytokeratin 18 may be associated with proliferation of human RPE both in vitro and in vivo.
Ki-67 detects a nuclear matrix-associated proliferation-related antigen: II. Localization in mitotic cells and association with chromosomes
Authors: Verheijen R. , Kuijpers H. J. H. , Driel R. van , Beck J. L. M. , Dierendonck J. H. van , Brakenhoff G. J. , Ramaekers F. C. S.
Published: April 2021

In interphase cells the proliferation-associated antigen recognized by monoclonal antibody Ki-67 is almost exclusively located in the nucleoli. When cells at several stages of mitosis were examined for the localization of the Ki-67 antigen, a striking redistribution could be observed. During prophase the distinct nucleolar Ki-67 fluorescence changed to a bright irregular meshwork throughout the nucleoplasm. At metaphase the antigen appeared to be distributed in a reticulate structure surrounding the condensed chromosomes, while at late telophase a punctated staining of the entire nucleoplasm was observed, which preceded the typical nucleolar localization pattern in each of the two daughter cells. Immunolabelling with Ki-67 of metaphase chromosome spreads revealed a circumferential staining of the individual chromosomes.

The Ki-67 antigen is preserved in nuclear matrix preparations obtained after in situ fractionation of interphase cells. When mitotic cells were exposed to such treatments, the obtained fluorescence data suggested that the antigen may be part of the chromosome scaffold.

Quantification of the Ki-67 fluorescence signal using flow cytometry revealed the highest staining intensities in mitotic cells. Furthermore, it was shown that nutritionally deprived cells became negative for Ki-67.
Intermediate filament collapse is an AtP-dependent and actin-dependent process
Authors: Hollenbeck P. J. , Bershadsky A. D. , Pletjushkina O. Y. , Tint I. S. , Vasiliev J. M.
Published: April 2021

In this study, we have investigated the properties of intermediate filament rearrangements using experimentally induced collapse of vimentin intermediate filaments in mouse fibroblasts. In these cells, depolymerizing microtubules by colchicine or vinblastine treatment at 37 °C results in a two-stage collapse of intermediate filaments. First, the vimentin filaments aggregate into large cables; then, the cables coil into a dense mass surrounding the nucleus. By using inhibitors of oxidative phosphorylation along with glucose deprivation to lower intracellular ATP levels by 95 %, we have found that both stages of intermediate filament collapse require ATP. However, once collapse has occurred, only the second stage can be reversed in the absence of microtubules by lowering ATP levels. An additional difference between the two stages of collapse was revealed by treating cells with cytochalasin D: the formation of intermediate filament cables still occurs after disruption of the actin filament system by cytochalasin, but the subsequent coiling of cables to form a perinuclear mass is strongly inhibited by these conditions, and can be reversed by applying cytochalasin to cells in which intermediate filaments have already undergone complete collapse. We propose that the formation of vimentin cables involves a phosphorylation event, while the coiling of cables into a perinuclear mass relies on interaction of intermediate filaments with a component of the actin cortex.
Modulation of melanoma cell adhesion to basement membrane components by retinoic acid
Authors: Edward Michael , Gold Jean A. , Mackie Rona M.
Published: April 2021

The effect of retinoic acid pretreatment on metastatic B16 melanoma cell adhesion in serum-free medium to tissue culture plastic precoated with fibronectin, laminin/nidogen, type I and type IV collagen was examined. Both control cells grown to subconfluence and cells treated with 10−6 M-retinoic acid adhered and spread rapidly on fibronectin (>75% following 1 h of incubation) but adhered poorly to type I collagen (<15%). Control cells adhered to laminin/nidogen (>35%), type IV collagen (>58%) and type IV collagen plus laminin/nidogen (>80%), while retinoic acid-treated cells showed a reduced ability to attach and spread on these substrata, the number of adherent cells being reduced by 61% on laminin/nidogen, by 19% on type IV collagen, and by 41% on type IV collagen plus laminin/nidogen following Ih of incubation. The minimum concentration of retinoic acid required to yield an effective reduction in adhesion was 10−7M for type IV collagen and 10−10M for laminin/nidogen. Melanoma cells harvested at low density showed a reduced adhesion to laminin/nidogen and type IV collagen compared to that of subconfluent control cultures, but also showed a reduced adhesion to fibronectin. The effect of retinoic acid on cell adhesion was not, however, due to reduced cell density, as the cells were seeded so that control and retinoic acid-treated cultures were of a similar density when harvested.
An immunocytochemical study of the proliferating cell nuclear matrix antigen p125/6.5 during rat spermatogenesis
Authors: Hadjiolova K. V. , Martinova Y. S. , Yankulov K. Y. , Davidov V. , Kancheva L. S. , Hadjiolov A. A.
Published: April 2021

In previous studies of proliferating mammalian cells a pl25/6.5 nuclear matrix antigen displaying a marked increase in mitotic cells has been identified. This antigen was investigated by immunocytochemistry of cryosections of testes at different stages of postnatal development: newborn, 20 days after birth and sexually mature rats. In Sertoli cells, the distribution of the p 125/6.5 antigen parallels [3H]thymidine incorporation: present in newborn and absent in sexually mature testes. The pl25/6.5 antigen is present also in some prespermatogonia of the newborn rat testis, which do not incorporate [3H]thymidine. At later stages of development, the pl25/6.5 antigen is present also in first meiotic prophase spermatocytes displaying an extrachromosomal nucleoplasmic distribution, while absent in spermatids and spermatozoa. These results show that the pl25/6.5 antigen increases not only during mitosis, but also during meiosis. They suggest further that this antigen is characteristic of both proliferating cells and cells (prespermatogonia) committed to proliferation.
Ontogenetic expression of the murine erythrocyte glycophorins
Authors: Dolci Elizabeth D. , Palade George E.
Published: April 2021

The major sialoglycoproteins or glycophorins of the murine erythrocyte membrane, gp2 (AÇ44000) and gp3 (Mr 29 000), are expressed as identical or closely related antigens by the three erythroid cell lines that succeed each other in normal mouse development. The embryonic forms of gp2 and gp3 differ from the adult forms by their mobility on SDS-PAGE. The apparent Mr values are increased by 1000–2000 for gp2 and 500 for gp3. An increase in the microheterogeneity of both embryonic and fetal forms of gp2 is also detected. Variations in glycosylation account in part for the apparent differences in Mr. Sizing of O-glycosidically linked oligosaccharide chains reveals that fetal glycophorins contain predominantly trisaccharide units while their adult counterparts are mostly tetrasaccharides. The kinetics of gp2 and gp3 biosynthesis in fetal liver are comparable to those established for the splenic erythroblasts of adult anemic mice. Like their adult counterparts, fetal glycophorins incorporate [3H] palmitate and [3H]galactose. The results indicate that, in murine ontogeny, distinct but antigenically related sialoglycoproteins are produced at each erythropoietic stage.
Development of the quadripolar meiotic apparatus in Funaria spore mother cells: analysis by means of anti-microtubule drug treatments
Authors: Busby C. H. , Gunning B. E. S.
Published: April 2021

Microtubule-dependent processes in Funaria hygrometrica spore mother cells (SMCs) were analysed by monitoring the effects of colchicine and oryzalin on pre-meiotic and meiotic events. The techniques used were electron microscopy, immunofluorescence microscopy of microtubules (MTs) and continuous video recording of events in treated and recovering live cells sampled at various stages of sporogenesis. Inferences drawn from previous work that the SMC plastids serve as MT-organizing centres were confirmed in so far as MT recovery in MT-depleted cells starts at the tips of the plastids. The MTs that emanate from these regions are required for positioning the plastids in the tetrahedral conformation, which defines the meiotic poles, for positioning lipid droplets in clusters at these poles and for positioning and holding the nucleus in the tetrahedral cage. If released, the nucleus can be moved by a non-MT system. Other phenomena not controlled by MTs are plastid elongation, maintenance of the tetrahedral conformation when the MTs are absent (during divisions or as a result of drug treatment) and (probably) development of the organelle band that spans the cell between divisions I and II.

In cells treated during division, when there is no nuclear envelope, the pattern of MT recovery is different: the plastids are inactive as microtubuleorganizing centres (MTOCs) but MTs reappear among the chromosomes. Spindles capable of transporting chromosomes regenerate. However, the importance of interactions between nucleus and plastids is highlighted by cases in which treatment has resulted in: (1) movement of the nucleus out of the quadripolar plastid cage; and (2) loss of the MTs at plastid tips that normally contribute to the spindle poles; in such cases quadripolarity is lost even though functional spindles return. Plastid MTOC activity returns when the nuclear envelope is in place, i.e. in interkinesis and after telophase II.
Distribution of a thiophosphorylated spindle midzone antigen during spindle reactivation in vitro
Authors: Wordeman Linda , Masuda H. , Cande W. Z.
Published: April 2021

Mitotic spindles isolated from the diatom Stephano-pyxis furris will elongate in vitro in the presence of ATP with a concurrent decrease in the width of the zone of microtubule overlap. A spindle-associated phosphoprotein that co-localizes with the zone of microtubule overlap in isolated spindles serves as a convenient marker for midzone-associated proteins other than microtubules. We have used a monoclonal antibody that labels this protein when it is artificially thiophosphorylated and studied its redistribution during spindle reactivation in vitro. As the spindle elongates midzone label accumulates in a successively narrower and brighter ring at the spindle midpoint with increasing time in ATP. Biotinylated bovine microtubule segments polymerized onto the ends of the diatom microtubules increase the overall width of the zone of microtubule overlap and serve as a marker for the boundary of the original diatom overlap zone. During elongation in ATP, the biotinylated segments move into the area marked by the monoclonal antibody, which does not decrease in width until the spindle has elongated to the point at which the zone of microtubule overlap delineated by the newly polymerized microtubules is smaller than the original overlap zone. We use these results to develop a model to explain the behaviour of nonmicrotubule midzone-associated proteins during spindle elongation.
Immunochemical localization of amiloride-sensitive sodium channels in sodium-transporting epithelia
Authors: Tousson Albert , Alley Charles D. , Sorscher Eric J. , Brinkley B. R. , Benos Dale J.
Published: April 2021

The localization of amiloride-sensitive Na+ channels in Na+-transporting epithelia was examined using antibodies made against amiloride-binding Na+ channel protein purified from bovine kidney. The distribution of the channel protein was determined in thick frozen sections at the light-microscopic level using indirect immunofluorescence, and at the electron-microscopic level using immunogold labelling. In the cells of both the intact bovine collecting tubule and A6 confluent monolayers, only the luminal or apical-facing surface membranes showed staining. Sodium channel proein was characteristically localized on microvillar domains of the apical μlasma membrane. Little or no basolateral membrane staining was evident. Channel protein was also absent from subapical vesicles and tight junctions, and was not found in bovine renal proximal tubules, cultured human secretory sweat coils, non-epithelial Chinese hamster ovary (CHO) cells or human skin fibroblasts.

Trypsinization of intact A6 monolayers prior to cell fixation abolished specific staining with antibody. Pretreatment with amiloride protected against this loss of staining. Thus, our probes are specific for amiloride-binding Na+ channel protein, and this channel protein is largely or comμletely confined to the apical membrane of Na+-transporting epithelia. The level and distribution of specific immunostaining in A6 cells was unchanged by aldosterone treatment, although channel activity, as measured by short-circuit current, increased threefold. This result demonstrates that Na+ channel protein is ever present at the cell surface and exists in both an active and an inactive form. We find no evidence that stimulation of Na+ uptake by aldosterone involves recruitment of new channels from a cytoμlasmic pool.
Patterns of expression of keratin 17 in human epithelia: dependency on cell position
Authors: Troyanovsky S. M. , Guelstein V. I. , Tchipysheva T. A. , Krutovskikh V. A. , Bannikov G. A.
Published: April 2021

By immunomorphology, using keratin 17-specific monoclonal antibody, it has been shown that this keratin is expressed only in the basal cells of a group of complex epithelia: glandular epithelium with myoepithelial component, transitional and pseudostratified epithelia. Immunolocalization of keratin 17 provides evidence that the expression of this keratin strongly depends on the cell position within epithelial structures. The topographical character of the keratin expression suggests that these proteins may be implicated in the generation of spatial organization of epithelial tissues.
Parameciumtrichocysts isolated with their membranes are stable in the presence of millimolar Ca2+
Authors: Lima Oscar , Gulik-Krzywicki Tadeusz , Sperling Linda
Published: April 2021

We have developed a simple and rapid procedure for the isolation of a pure fraction of Paramecium trichocysts (mature secretory vesicles) with their membranes. Since in wild-type Paramecium cells essentially all trichocysts are docked at pre-formed cortical sites, trichocysts were isolated from cells in which functional trichocysts remain free in the cytoplasm owing to a mutation, tam6, that affects the docking site. Examination of the preparations by freeze-fracture electron microscopy confirms the presence of the membranes. The distribution of particles in the membranes of the isolated trichocysts and in the membranes of wild-type trichocysts in situ are nearly identical and this argues against any rearrangement of the membranes during the isolation procedure. Although the trichocyst matrix undergoes a dramatic structural transition in the, presence of Ca2+ and water (matrix expansion), the isolated vesicles with intact membranes are perfectly stable in the presence of millimolar free Caz+. This result supports a chronology in which the first step in exocytosis is membrane fusion, the swelling of vesicle contents occurring only afterwards, once the contents come into contact with the water and Ca2+ of the external medium. The role of swelling would then be to help disperse, propel or otherwise empty the contents of the vesicle outside the cell.
Immunocytochemical detection of non-histone nuclear antigens in cryosections of developing somatic embryos from Daucus carota L
Authors: SANCHEZ-PINA M. A. , KIEFT H. , SCHEL J. H.N.
Published: April 2021

The distribution of non-histone nuclear antigens was studied during early somatic embryogenesis of carrot using nuclear antibodies specific for small nuclear RNPs (snRNPs), nuclear matrix proteins and nucleolar proteins. Immunofluorescence showed that in interphase cells snRNPs and nuclear matrix proteins were localized in the nucleus with a speckled pattern. Immunoelectron microscopy indicated that the snRNPs were irregularly distributed while the nuclear matrix proteins showed a distribution preferentially over the condensed chromatin. None were localized in the nucleolus. The nucleolar proteins were localized in both dense fibrillar and granular components while the fibrillar centres were devoid of gold particles.

The localization of these antibodies in plant cell nuclei, in a way comparable to the patterns described in animal cells, indicates the general presence of snRNPs in all eukaryotic nuclei. It further confirms the relation of the nuclear matrix proteins to plant cell proliferation events and the possible role of the nucleolar proteins in both rRNA transcription and preribosome assembly.
Enolase exists in the fluid phase of cytoplasm in 3T3 cells
Authors: Pagliaro Len , Kerr Karen , taylor D. Lansing
Published: April 2021

We have investigated the intracellular distribution and mobility of the glycolytic enzyme enolase, using functional fluorescent analogs labeled with the succinimidyl esters of carboxyfluorescein (Flenolase) and carboxytetramethylrhodamirie (Rh-enolase). In contrast to aldolase, neither native enolase nor labeled enolase gelled filamentous actin (F-actin), as measured by falling-ball viscometry, indicating a lack of interaction between enolase and F-actin. Fluorescence redistribution after photobleaching (FRAP) measurements of the dflfusion coefficient (D) of Fl-enolase in aqueous solutions gave a value of D337,aq = 6·08×10−7cm2s-1, and no immobile fraction, consistent with a native molecular weight of 90 000. These values were not significantly dHferent with Rh-enolase, or in the presence of F-actin, 2-ρhosρhoglycerate or F-actin–aldolase gels, demonstrating that neither Fl-enolase nor Rh-enolase binds to F-actin or aldolase in vitro. FRAP measurements of Fl- and Rh-enolase microinjected into living Swiss 3T3 cells revealed spatial differences in the diffusion coefficient, but not the mobile fraction. In the perinuclear cytoplasm, we measured an apparent diffusion coefficient of l·1×10−7cm2s-1, compared to 7·1×10−8cm2s-1 in the peripheral cytoplasm, with ≈ 100% mobility of Fl- or Rh-enolase in both regions. Imaging of cells co-injected with Rh-enolase and size-fractionated FITC–dextran (FD-90) revealed that Rh-enolase entered the nucleus, while FD-90 was excluded. Ratio imaging showed a relatively high nuclear ratio of Rh-enolase/FD-90, and a uniform cytoplasmic ratio, with no indication of increased concentration of enolase around stress fibers. These data demonstrate that Rh- and Fl-enolase do not bind to F-actin in vitro, and are 100% mobile in vivo. Together with our recent finding that a significant fraction of aldolase binds to F-actin in vivo and is immobile in vivo, these data suggest a correlation between actin-binding activity and cytoplasmic mobility of glycolytic enzymes.
Type v collagen synthesis and deposition by chicken embryo corneal fibroblasts in vitro
Authors: Mclaughlin Jacqueline Shea , Linsenmayer Thomas F. , Birk David E.
Published: April 2021

Chick embryo corneal fibroblasts were grown in culture to study the processes whereby fibroblasts regulate the deposition and organization of the collagenous, secondary stroma. The effects of an existing type I collagen substratum, cell density, and serum concentration on type V collagen synthesis were investigated. Type V collagen represented approximately 20% of the total fibrillar collagen synthesized, regardless of whether the cells were subcultured, grown on untreated or collagen-coated plastic, grown under confluent or subconfluent conditions, or grown in the presence of low (0.1%) or high (10.0%) serum concentrations. The synthesis of type V collagen remained constant at 20% of the total collagen when cells were grown in 1.0% serum, even though total collagen synthesis increased nearly twofold when compared to total synthesis in 0.1% or 10.0% serum. Immunocytochemistry with anti-collagen, type-specific monoclonal antibodies revealed a homogeneous population of cells synthesizing types I and V collagen. The fibrils deposited by cells grown in a three-dimensional collagen matrix contained a helical epitope on the type V molecule that was inaccessible unless the fibrillar structure was disrupted, mimicking the situation in situ. The production in vitro of heterotypic fibrils, with a constant l/V ratio and molecular packing mimicking the natural stroma, offers opportunities for studying in more detail this important process, which is essential for optical transparency.
Expression of oncomodulin does not lead to the transformation or immortalization of mammalian cells in vitro
Authors: Mes-Masson Anne-Marie , Masson Sylvie , Banville Denis , Chalifour Lorraine
Published: April 2021

A recombinant plasmid (pMTONCO) containing the coding sequences for rat oncomodulin under the direction of the metallothionein promoter was constructed. pMTONCO was co-transfected with the pSV2-NEO plasmid into primary mouse kidney cells or Rat-1 cells using the calcium phosphate technique and stable transformants were isolated after selection with G418. Transcription from the metallothionein promoter was inducible with heavy metals and produced an oncomodulin-specific mRNA. The presence of oncomodulin protein in stable cell lines was verified by immune-precipitation with specific antisera. While a plasmid encoding the polyomavirus T-antigens was able to prolong the life-span of primary mouse kidney cells in culture, no equivalent activity was noted when the pMTONCO plasmid was used to transfect primary cells. When expressed in Rat-1 cells, oncomodulin did not affect the growth properties of these cells, nor did it predispose cells to higher frequencies of oncogenic transformation to a viral oncogene. We conclude that oncomodulin is neither an immortalizing nor transforming agent in vitro.
Dynamics of cytoplasmic organelles in the cell cycle of the fission yeast Schizosaccharomyces pombe: three-dimensional reconstruction from serial sections
Authors: Kanbe Toshio , Kobayashi Issei , Tanaka Kenji
Published: April 2021

Changes in the ultrastructure of the fission yeast Schizosaccharomyces pombe during the cell division cycle were analyzed by three-dimensional reconstruction of serial section electron micrographs of freeze-substituted cells. Cytoplasmic vesicles were found at the cell ends during interphase and at the equatorial zone of cells undergoing cytokinesis. Filasomes behaved in a similar but temporally retarded way to vesicles. Microfila-ment(mf)-associated granules were found attached to the plasma membrane at the growing ends. Microfilaments were identified against the plasma membrane and adjacent to developing septa. From these observations it is suggested that mf-associated structures such as filasomes constitute dense knotsof actin network that function in localized cell wall growth by controlling the deposition of cytoplasmic vesicles. Dictyosomes occur as tubular and fenestrated cisternae with associated cytoplasmic vesicles. They were distributed uniformly in the cytoplasm and did not change significantly during the cell cycle. Changes in the three-dimensional localization of cytoplasmic microtubules and mitochondria are also described.
Human macrophage-mediated cytotoxicity of Schistosoma mansoni functional and structural features of the effector cells
Authors: Cottrell B. J. , Pye C. , Glauert A. M. , Butterworth A. E.
Published: April 2021

Human monocytes were purified from peripheral blood and cultured in vitro on hydrophobic membranes. Such cells developed into mature tissuetype macrophages after approximately 1 week in culture. During this maturation period the macrophages developed a potent cytotoxic mechanism whereby they could kill the schistosomula of Schistosoma mansoni in standard in vitro cytotoxicity assays. Cytological and ultrastructural studies of the cells grown in vitro indicated that macrophages developed many of the classical histological and ultrastructural features of‘activated’ cells with ruffled plasma membranes and significant increases in rough endoplasmic reticulum and Golgi vesicles. Effective cytotoxicity depended upon contact of the effector cells and their parasite target. Further, experiments using metabolic inhibitors indicated that cytotoxicity was dependent upon protein synthesis. Initial results point to the macrophage factor being distinct from some of the better-characterised macrophage secretory products such as tumour necrosis factor, proteases and products of oxygen metabolism.
Suppression of malignancy in human lymphoid cell hybrids: The role of differentiation
Authors: Saltman D. , Ross J. A. , Gordon J. M. , Krajewski A. S. , Ross A. R. , Foster M. E. , Steel C. M.
Published: April 2021

Human somatic cell hybrids were generated from fusions between a tumorigenic B-cell line, and mononuclear cells from a patient with low-grade B-cell lymphoma in which the neoplastic cells largely corresponded to cells at an intermediate stage of differentiation. The resulting hybrids and the parent cells were immunophenotyped, karyotyped and genotyped in an effort to determine whether the stage of B-cell development of the fusion partners was important in the suppression of malignancy. The majority of hybrids demonstrated suppression of tumorigenicity as measured in immunodeprived mice. Chromosome markers and immunoglobulin gene rearrangements found in the patient’s neoplastic cells were also present in several of the hybrids. One of the tumorigenic hybrids retained specific chromosomes also found in the non-tumorigenic hybrids, but demonstrated an immunophenotype and genotype possibly associated with a subset of early B-cells. In the non-tumorigenic hybrids that retained most of the chromosomes from both parents there was evidence of plasmacytoid differentiation. These results suggest that the ability of B-lymphocytes to suppress malignancy in human lymphoid hybrids may be dependent on the corresponding stage of normal B-cell ontogeny, and that suppression is associated with differentiation.