bluuebunny/crossref_metadata_2025 huggingface dataset:
Isoproterenol downregulation of statin-related gene expression in the rat parotid gland
Authors: Ann David K. , Wechsler Aharon , Lin H. Helen , Wang Eugenia
Published: April 2021

Statin, a 57 kilodalton (kDa) nuclear protein, is characteristically found in nonproliferating cells in culture as well as nondividing cells of a wide range of highly differentiated tissues. Moreover, cells in culture that are statin positive lose this statin expression when re-entering the cell-cycle traverse. In this work, statin expression was investigated in the parotid gland of untreated rats and those treated with isoproterenol (IPR), a proliferation-inducing catecholamine. Indirect immunofluorescence microscopy revealed specific nuclear staining with antistatin monoclonal antibody (S-44) in the acinar and ducts cells of the untreated rats but significantly reduced in those induced with isoproterenol. To characterize the protein recognized by S-44, protein extracts from both tissues were immunoblotted and incubated with S-44. The antibody reacted specifically with a 48 kDa protein in the extract of the parotid glands from untreated rats while no reaction was detected in that of the proliferation-induced ones. These observations along with the result that a statin-like (SI) transcript is downregulated by isoproterenol in the parotid glands further support the notion that the disappearance of statin-related expression is associated with the IPR-induced proliferation in the rat parotid glands. The discrepancy between the apparent molecular mass of the protein identified by S-44 in nonproliferating parotid cells and that of statin originally found in fibroblasts, suggests that either a modified form of statin may be present in the parotid gland, or this 48 kDa protein may be a member of the nonproliferative statin-like family.
Dominance of a NOR (nucleolar organizer region) over its allele and over its sister NOR after asymmetric 5-azacytidine substitution in plant chromosomes
Authors: Torre C. De La , Gimenez-Abian J. F. , Gonzalez-Fernandez A.
Published: April 2021

Dominance of a 5-azacytidine-substituted nucleolar organizer region (AZA NOR) over another with normal DNA when confined to a common nucleus or to different nuclei in a common cell was studied in Allium cepa L. root meristems. AZA administration took place for one S period. The assessment of NOR dominance was done either in early Gi (nucleologen-esis) or later in interphase (mature nucleoli). At the mitosis immediately preceding the second interphase after AZA, the segregated AZA and normal chromatids of each chromosome of the pair of homologues were confined to a single binucleate cell by inhibiting the formation of the cell plate. Anaphase segregation of the homologous AZA NORs was seen to occur at random.

The AZA NORs completed nucleologenesis after a shorter time than normal NORs, independently of the intranuclear or intracellular presence of other NORs. After nucleologenesis was over in the AZA NORs, a few of the normal NORs, when in a common nucleus, remained permanently inefficient in developing a full-size mature nucleolus or any nucleolus at all (partial or total dominance, respectively).

Dominance of an AZA NOR over its sister or allelic NOR with normal DNA led to the new nucleolar patterns of asymmetry and inverse symmetry, respectively, making their appearance in these binucleate cells.
How relevant is the Escherichia coli uvrabc model for excision repair in eukaryotes?
Authors: Hoeijmakers J. H. J.
Published: April 2021

Knowledge about the DNA excision repair system is increasing rapidly. A detailed model for this process in Escherichia coli has emerged in which a lesion in the DNA is first recognized by the UvrA2B helicase complex. Subsequently, UvrC mediates incision on both sites of the DNA injury. Finally, the concerted action of helicase II (UvrD), polymerase and ligase takes care of removal of the damage-containing oligonucleotide, DNA resynthesis and sealing of the residual nick.

In the eukaryotes, yeast and mammals a total of 10 excision repair genes have been analysed thus far. However, little is still known about the molecular mechanism of this repair reaction. Amino acid sequence comparison suggests that at least three DNA helicases operate in eukaryotic nucleotide excision. In addition, a striking sequence conservation is noted between human and yeast repair proteins. But no eukaryotic homologs of the UvrABC proteins have been identified. In this Commentary the parallels and differences between the prokaryotic and eukaryotic excision repair pathways are weighed in an attempt to assess the relevance of the E. coli model for the eukaryotic system.
The membrane skeleton of Pseudomicrothorax: I. isolation, structure and composition
Authors: Peck Robert K. , Duborgel FranÇoise , Huttenlauch Irm , De Haller Gerard
Published: April 2021

The epiplasm membrane skeleton of the ciliated protozoan Pseudomicrothorax dubius has been isolated and its three-dimensional structure and constituent proteins have been examined. The epiplasm functions as a cytoskeleton to define cell shape and the position of some cortical organelles. Scanning electron microscopy of the isolated epiplasm reveals a rococo cytoarchitecture in which basal bodies and trichocyst attachment sites are arranged in precise geometric arrays. SDS-PAGE reveals 40 bands, one of which is quantitatively the major band of the epiplasm and is composed of at least 3 different proteins and numerous isoelectric variants, as revealed by two-dimensional electrophoresis and peptide mapping. Polyclonal antisera were produced against native (antiserum 15) and SDS-denatured (antiserum 18) epiplasm. On immunoblots, antiserum 15 labels the hydrophilic proteins that are extracted from the epiplasm by treatment with dilute acid solution and that are predominantly glycoproteins, four of which are labeled with Concanavalin A on Western blots. On Lowicryl thin sections, antiserum 15 labels the epiplasm uniformly, except for the terminal plates, indicating that the glycoproteins are integral components of the epiplasm and are not membrane contaminants in the epiplasm fraction. Concanavalin A labeling of Lowicryl sections supports the latter result. On immunoblots, antiserum 18 labels the acid-insoluble epiplasm bands, the major structural elements of the epiplasm. One of the epiplasm bands at 52×103Mr is labeled by an anti-/) tubulin monoclonal antibody. Evidence is presented that this fi tubulin is not due to microtubule contamination of the epiplasm fraction.
Freeze substitution reveals a new model for sporangial cleavage inPhytophthora, a result with implications for cytokinesis in other eukaryotes
Authors: Hyde G. J. , Lancelle S. , Hepler P. K. , Hardham A. R.
Published: April 2021

Rapid freezing and freeze substitution (RF-FS) have been used to re-examine the process by which the multinucleate sporangium of the Oomycetes, Phytophthora cinnamomi and P. palmivora, is subdivided into uninucleate zoospores. The results indicate a new model for sporangial cleavage in Phytophthora and suggest that the currently accepted model is based on interpretation of artefacts caused by chemical fixation. The previous model describes cleavage as a two-stage process in which specialized cleavage vesicles first become positioned at the boundaries of each future subdivision and later fuse to compartmentalize the sporangium. RF-FS, however, indicates that cleavage results from the progressive extension of paired sheets of membrane along the future subdivision boundaries. These sheets finally interconnect and subdivide the sporangium. Cleavage vesicles are only evident in preliminary stages of this process and are never aligned along the future boundaries, contrary to the observations of studies based on chemical fixation. Chemical fixation apparently causes the membranous sheets to vesiculate, even at relatively advanced stages of cleavage, thus giving the misleading impression that the resulting network of lined-up vesicles is an intermediate stage in the cleavage process. This finding has wide-ranging implications for the understanding of eukaryotic cytokinesis, because all previous studies that describe vesicle alignment and fusion have relied upon chemical fixation. Other novel features revealed by RF-FS include an extensive extracellular matrix within the sporangium that could be involved in zoospore release, and a trans-Golgi network.
Analysis of human mitochondrial transcripts using electron microscopic in situ hybridization
Authors: Escaig-Haye FranÇoise , Grigoriev Vladimir , Peranzi Gabriel , Lestienne Patrick , Fournier Jean-Guy
Published: April 2021

Human mitochondrial transcripts have been examined at the ultrastructural level. After contact with ultrathin sections of a human lymphoid cell line (CEM) embedded in Lowicryl K4M, biotinylated mitochondrial probes yield specific hybrids identified by a colloidal gold immunocytochemistry marker that visualizes rRNA and mRNA coding for respiratory chain polypeptides CO II, CO III and ATPase-6. The mitochondrial transcripts are preferentially located close to the inner membrane, particularly the cristae, suggesting that intra-organelle protein synthesis is intimately associated with the mitochondrial membrane system. Quantitative analysis indicates that the mitochondria concentrate the labeling with intensities that vary with the type of RNA and that the nucleus induces a fight hybridization signal with each mitochondrial probe. The visualization of human mitochondrial DNA expression in correlation with the fine anatomy of the mitochondria constitutes a new approach for fundamental research on the organelle and for analyzing its behaviour in human mitochondrial diseases.
The timing of the formation and usage of replicase clusters in s-phase nuclei of human diploid fibroblasts
Authors: Kill Ian R. , Bridger Joanna M. , Campbell Keith H. S. , Maldonado-Codina Gabriela , Hutchison Christopher J.
Published: April 2021

The sites of nascent DNA synthesis were compared with the distribution of the proliferating cell nuclear antigen (PCNA) in S-phase nuclei of human diploid fibroblasts (HDF) by two in vitro techniques. Firstly, proliferating fibroblasts growing in culture that had been synchronised at S-phase were microinjected with the thymidine analogue biotin-ll-dUTP. The sites of incorporation of biotin into injected cells were compared with the distribution of PCNA by indirect immunofluorescence microscopy and laser scanning confocal microscopy (LSCM). In common with other studies, a progression of patterns for both biotin incorporation and PCNA localisation was observed. However, we did not always observe coincidence in these patterns, the pattern of biotin incorporation often resembling the expected, preceding distribution of PCNA. In nuclei in which the pattern of biotin incorporation appeared to be identical to the distribution of PCNA, LSCM revealed that not all of the sites of PCNA immunofluorescence were incorporating biotin at the same time. Secondly, nuclei which had been isolated from quiescent cultures of HDF were innoculated into cell-free extracts of Xenopus eggs which support DNA replication in vitro. Following innoculation into these extracts DNA replication was initiated in each nucleus. The sites of DNA synthesis were detected by biotin-ll-dUTP incorporation and compared with the distribution of PCNA by indirect immunofluorescence. Only a single pattern of biotin incorporation and PCNA distribution was observed. PCNA accumulated at multiple discrete spots some 15 min before any biotin incorporation was observed. When biotin incorporation did occur, LSCM revealed almost complete coincidence between the sites of DNA synthesis and the sites at which PCNA was localised.
Dictyostelium discoideum mutants resistant to the toxic action of methylene diphosphonate are defective in endocytosis
Authors: Bof Mireille , Brénot Françoise , Gonzalez Carlos , Klein Gérard , Martin Jean-Baptiste , Satre Michel
Published: April 2021

Methylene diphosphonate is taken up in Dictyostelium discoideum amoebae by fluid-phase pinocytosis, and it inhibits growth through the production of methylene analogs of adenosine triphosphate and diadenosine tetraphosphate. Methylene diphosphonate resistance was thus used as the basis of a screening strategy for the isolation of endocytosis mutants. Fifteen Dictyostelium mutants, whose growth was resistant to 7.5 mM methylene diphosphonate, were obtained and three of them were characterized in more detail. They were partially defective in fluid-phase pinocytosis (both the rate and extent of FITC-dextran entry were reduced to 40-50% of the parent type activity) and they had smaller amounts of several lysosomal enzymes, such as acid phosphatase, N-acetylhexosaminidase, a-mannosidase (20-60% of the parent type activities). In contrast to the lysosomal hydrolases, the mutants had unchanged activities for enzyme markers selective for other compartments. They appeared phenotypically similar to the Dictyostelium mutant HMW570, which is defective in fluid-phase pinocytosis and oversecretes lysosomal enzymes. The methylene diphosphonate-resistant mutants were found to be unable to acidify fully their endosomal compartments and they have an increased endosomal pH, as shown by the use of the pH-sensitive fluorescence of FITC-dextran. Furthermore, the hypothesis proposing a defective acidification of the endosomal pathway was supported by the measurement of A TP-dependent vesicular acidification with acridine orange, and by in vivo 31P NMR spectroscopy with aminomethylphosphonate as a pH probe.
The β4 subunit of the integrin family is displayed on a restricted subset of endothelium in mice
Authors: Kennel S. J. , Godfrey V. , Ch’ang L. Y. , Lankford T. K. , Foote L. J. , Makkinje A.
Published: April 2021

More than 15 subunits of the integrin family of cell surface adhesion molecules have been identified. The α6β4 integrin has recently been identified as a component of hemidesmosomes of stratified squamous epithelium. The monoclonal antibody (mAb) 346-11A binds to the β4 subunit in mice. Sequence analysis of a cDNA clone coding for this epitope localizes reaction of the mAh to a portion about half way through the extracellular domain at the beginning of the cysteine-rich region.

Sites of β4 expression in mice were detected by autoradiographic analysis of tissues collected from mice 24 or 48 h after intravenous injection of 125I-labeled mAh 346-11A. This non-quantitative technique emphasizes detection of antigen exposed in the vascular space.

These data show that in addition to the epithelia of several organs, the endothelia of intermediate vessels throughout the body are sites of β4 expression. In particular, endothelium of larger vessels but not capillaries in lung, vessels in thymus, spleen and Peyer’s patches, and portal vessels but not the central veins of the liver, are positive. The expression of the fβ4 integrin in blood vessels may indicate a specialized function for β4 at these sites that is distinct from its role in hemidesmo-some-mediated attachment.
Distinct distribution of vimentin and cytokeratin in Xenopus oocytes and early embryos
Authors: Torpey N. P. , Heasman J. , Wylie C. C.
Published: April 2021

We report the identity of a major component of Tritoninsoluble extracts from Xenopus oocytes and early embryos. In a previous paper we showed that an antibody, Z9, cross-reacts with two polypeptides from such extracts (Mr 56,000 and 57,000) as well as Xenopus vimentin. Direct microsequencing of the Mr 57,000 protein shows near identity of three tryptic fragments with regions of the predicted amino acid sequence of XCK1(8), a basic cytokeratin whose mRNA is known to be expressed in Xenopus oocytes. We have raised an antibody, CK7, against a fusion protein generated from this cDNA. The specificity of this antibody has been tested using 1- and 2-dimensional immunoblotting, which show that it is specific for the Mr 56,000 and 57,000 proteins, suggesting that these two proteins may be the products of two non-allelic XCK1(8) genes. The antibody does not cross-react with vimentin. We have used CK7 to follow the distribution of XCK1(8) throughout development by immunoblotting and immunocytochemistry. In larval stages, strong staining is seen in the notocord, the apical epithelia of the gut, the mesentery, and a few cells in the spinal cord. In oocytes and early embryos, two distinct intermediate filament (IF) networks can be distinguished: a cortical cytokeratin network, and a deeper vimentin one. In addition, the oocyte germ plasm stains with Z9 but not CK7. We propose that such distinct distributions of each IF protein reflect functional differences during early development.
Mesenchyme-mediated effect of dexamethasone on laminin in cocultures of embryonic gut epithelial cells and mesenchyme-derived cells
Authors: Simo P. , Simon-Assmann P. , Arnold C. , Kedinger M.
Published: April 2021

Previous studies have shown that glucocorticoids accelerate intestinal maturation and that this process is mediated by the mesenchymal cells. The possible involvement of laminin (LN), a basement membrane component, in this mesenchymal mediation has been analyzed. For this purpose, the influence of dexamethasone (DX) on the synthesis of LN, its chain composition and its cellular distribution has been examined biochemically and immunocytochemically in two different mesenchyme-derived cell populations, fetal intestinal mesenchymal cells and fetal skin fibroblasts, as well as in cocultures of intestinal endodermal cells seeded on top of confluent fetal skin fibroblasts. Neither the amount of metabolically labeled LN purified by affinity chromatography (expressed per mg cell proteins), nor the A versus B chain ratio monitored after separation on gel electrophoresis and immunoblotting, showed significant differences after 5 days of DX treatment. However, glucocorticoids induced a shift from secreted to cell-associated LN molecules paralleling a striking difference in the immunostaining pattern of intracellular and surface LN in the mesenchyme-derived cell monocul-tures; the granular intracytoplasmic LN staining in the control cultures was replaced by a fibrillar organization of LN molecules concomitantly with an increased accumulation at the cell surface. In 2-day DX-treated cocultures, there was an acceleration of LN deposition at the epithelial-fibroblastic interface, which accompanied the enhanced expression of epithelial cell differentiation markers (brush border digestive enzymes). These DX-induced changes can be blocked by the addition of antiLN antibodies in the culture medium.

These findings further support the concept that glucocorticoid action on intestinal epithelial cells involves alterations in the extracellular microenvironment, assessed here for LN molecules, occurring at the level of the mesenchymal cell compartment. These changes may contribute to an accelerated organization of LN at the epithelial-mesenchymal interface and subsequently to epithelial differentiation.
Divalent cations and extracellular matrix receptor function during newt epidermal cell migration
Authors: Mahan James T. , Donaldson Donald J.
Published: April 2021

Skin expiants were placed in plastic dishes coated with fibronectin (FN), fibrinogen (FGN) or collagen. Explants were cultured for 16 h in serum-free medium containing calcium (Ca) and magnesium (Mg), or in medium containing either Ca-only, Mg-only or manganese (Mn)-only. In Ca/Mg, migrating kératinocytes on ail test substrata produced a sheet of contiguous cells that formed a robust halo around each explant. When Ca was the only divalent cation added, the halos in FN- and FGN-coated dishes were approximately 70-80% as large as in Ca/Mg. On collagen, however, the halos were significantly smaller than on the other two substrata. This substratum-specific response in Ca-only suggests that migration on collagen is fundamentally different than migration on FN and FGN. Halos as large or larger than those in Ca/Mg formed on all three substrata in Mg-only. In this case, the halos were not in the form of a sheet of contiguous cells, but were composed of dissociated cells that had migrated from the explant. Individual cells likewise migrated from explants cultured in medium containing Mn-only; however, these halos were never as large as in Mg-only. Thus, while exogenous Ca appears to be an absolute requirement for maintenance of cell-cell connections, the cell-substratum interactions that lead to migration can utilize either Ca, Mg or, to a lesser extent, Mn.

Additionally we found that migration on the generally nonpermissive protein, BSA, was not improved by the presence of Mn. Hence, even though Mn can mediate to some extent migration on FN, FGN and collagen, presumably by binding to integrin-like receptors on the cell surface, Mn does not alter receptor specificity in a way that permits migration on BSA.

To analyze the possibility that individual newt kératinocytes possess receptors for more than one ECM molecule, we cultured skin explants in Mg-only on a narrow zone of FN bordered on one side by collagen and on the other by uncoated plastic. Individual kératinocytes were able to migrate across the FN strip and continue on collagen but not on uncoated plastic, indicating that at least some kératinocytes have receptors for at least two matrix molecules.
Studies of muc-1 mucin expression and polarity in the mouse mammary gland demonstrate developmental regulation of muc-1 glycosylation and establish the hormonal basis for mrna expression
Authors: Parry Gordon , Li Jil , Stubbs John , Bissell Mina J. , Schmidhauser Christian , Spicer Andrew P. , Gendler Sandra J.
Published: April 2021

Muc-1 is a major mucin glycoprotein expressed on the surface of mammary epithelial cells. It has attracted considerable attention as it is expressed in an aberrant form on many breast tumor cells. Here we describe studies using a recently obtained cDNA probe of Muc-1 expression during lactogenic development in the mouse. Northern blot analysis demonstrated that Muc-1 is expressed at all stages of lactogenic development but its levels are increased very significantly during midpregnancy and into lactation. The basis of this was examined using CID-9 mammary epithelial cell cultures. It was found that in the presence of insulin Muc-1 mRNA levels were increased by both hydrocortisone and prolactin, with the combination of the three hormones supporting maximum expression. Muc-1 mRNA levels were also modulated by culturing cells on a basementmembrane-like extracellular matrix that promoted mRNA levels 5-to 10-fold above levels in cells cultured on plastic tissue culture dishes. Immunocytochemical studies using monoclonal antibodies to carbohydrate epitopes on Muc-1 demonstrated that while Muc-1 was found at all developmental stages, it became increasingly sialylated during the course of pregnancy and into lactation. Additionally, we found that while Muc-1 is tightly polarized to the apical surface of the epithelium of lactating and pregnant mice it exhibited a less-polarized distribution on a small proportion of ductal cells in virgin mice. We conclude that the expression of Muc-1 is regulated at several different levels and by a number of different factors. We speculate that this may reflect different functional roles for Muc-1 at different stages of mammary development.
The blood-brain barrier regulates the expression of a macrophage sialic acid-binding receptor on microglia
Authors: Perry V. H. , Crocker P. R. , Gordon S.
Published: April 2021

In vitro the expression of a sialic acid-binding receptor on murine macrophages, sialoadhesin, is regulated by exposure to an inducing agent present in serum. We have used immunocytochemistry to examine the macrophage populations of the nervous system in order to test whether this serum inducing agent (SIA) also regulates sialoadhesin expression in vivo and whether plasma proteins may influence the phenotype of macrophages of the nervous system. Microglia, the resident macrophages of the central nervous system, reside behind the blood-brain barrier and do not express sialoadhesin. Microglia and macrophage populations in the cicumven-tricular organs, choroid plexus and leptomeninges are exposed to plasma proteins and some macrophages express sialoadhesin at these sites. Injury to the CNS, which damages the blood-brain barrier, induces sialoadhesin expression on a proportion of macrophages and microglia within the parenchyma. The expression of sialoadhesin matches the temporal and spatial distribution of the plasma extravasation into the brain parenchyma.

These experiments show that exposure to SIA is necessary for sialoadhesin expression and lend further support to the idea that the phenotype of microglia is in part regulated by the presence of the blood-brain barrier.
The role of primer recognition proteins in DNA replication: association with nuclear matrix in Hela cells
Authors: Vishwanatha Jamboor K. , Jindalf Hitesh K. , Davis Randall G.
Published: April 2021

Primer recognition proteins (PRP) enable DNA polymerase a to utilize efficiently DNA substrates with low primer to template ratios. We have previously identified the protein-tyrosine kinase substrate annexin n, and the glycolytic enzyme 3-phosphoglycerate kinase as components of PRP. As a step towards elucidation of the role of PRP in the process of DNA replication, we have investigated the subcellular distribution and specific association of these proteins with the nuclear matrix in HeLa cells. Nuclear extracts prepared from HeLa cells in S phase contain the enzymatic activity of 3-phosphoglycerate kinase (PGK) and phospholipase A2 inhibitory activity of annexin II. Monomer annexin II is approximately equally distributed between the nuclear and cytoplasmic fractions, while a majority of PGK is in the cytoplasm. Immunoblot analyses reveal the presence of these two proteins in nuclei, specifically associated with the nuclear matrix. This is further confirmed by observation of the presence of annexin H and PGK in isolated nuclear matrices by immunoelectron microscopy. The phospholipase A2 inhibitory activity of annexin H colocalizes with the nuclear matrix-bound annexin II. A related protein, annexin I, is not detectable in the nuclear extracts and nuclear matrix. A slower-migrating (perhaps modified) form of annexin n is found to be associated with the nuclear matrix. Attempts to dissociate PGK and annexin H from the nuclear matrix with octyl-β-glucoside, high salt or metal ion chelators were unsuccessful, suggesting that the interaction is very strong.
cdc2 kinase-induced destabilization of map2-coated microtubules in Xenopus egg extracts
Authors: Faruki Shamsa , Dorée Marcel , Karsenti Eric
Published: April 2021

During the interphase to metaphase transition, microtubules are destabilized by a cdc2 kinase-dependant phosphorylation event. This destabilization is due to a dramatic increase in the rate at which each growing microtubule starts to shrink (catastrophe rate). In principle, this could be brought about by lowering the affinity of stabilizing MAPs for the microtubule wall, by activating a factor that would actively increase the catastrophe rate or by an alteration of both parameters. Here we examine the stabilizing effect of bovine brain MAP2 on microtubules assembled in interphase Xenopus egg extracts. We show that this MAP strongly stabilizes microtubules assembled in the extracts against nocod-azole-induced depolymerization. However, it does not protect them from the cdc2 kinase-induced shortening and destabilization. Moreover, the steady-state length of centrosome-nucleated microtubules in cdc2-treated extracts containing MAP2 is similar to that found in extracts lacking exogenous MAP2. We also show that although exogenous MAP2 is phosphorylated by cdc2 kinase in the extract, this is not the cause of microtubule destabilization. These results indicate that increased microtubule dynamics during mitosis is due to the activation of a factor that can function independently of the presence of active, stabilizing factors.
Inhibition of mitosis by okadaic acid: possible involvement of a protein phosphatase 2A in the transition from metaphase to anaphase
Authors: Vandré Dale D. , Wills Victoria L.
Published: April 2021

The effects of the protein phosphatase inhibitor okadaic acid were examined using the pig kidney cell line LLC-PK. At relatively low concentrations of the inhibitor (8–40 μM), cells became blocked in a metaphase-like mitotic state beginning 6–8 h after initial treatment. Spindle microtubules were present throughout the period of the mitotic block, but were not stabilized since they remained sensitive to nocodazole depolymerization. With increasing length of the mitotic block chromosome alignment at the metaphase plate was disrupted and multipolar spindles developed. Cells continued to accumulate in mitosis for at least 24 h, indicating that at these low concentrations okadaic acid was not cytotoxic, but rather acted as a cytostatic agent. Upon release of the okadaic acid block, mitotic LLC-PK cells recovered and completed anaphase. After extended periods of treatment some cells were able to escape the okadaic acid-induced mitotic block. These cells were multinucleate and had undergone cytokinesis in the absence of chromosome segregation. At higher concentrations of okadaic acid (0.5–1.0 μM), mitosis was blocked within 30-60 min of treatment. However, within 90–120 min treated cells rounded up and detached from the monolayer, regardless of whether they were in interphase or mitosis. Cytoplasmic microtubules were depolymerized in the detached cells, and these cells could not recover from the cytotoxic effects of such high concentrations of okadaic acid. Thus, differential effects of the phosphatase inhibitor could be demonstrated, depending upon the concentration of okadaic acid applied to the cultures. The okadaic acid-induced mitotic blockage was probably due to the inhibition of a type 2A protein phosphatase that is involved in the transition from metaphase to anaphase.
Cell cycle-specific disruption of the preprophase band of microtubules in fern protonemata: effects of displacement of the endoplasm by centrifugation
Authors: Murata Takashi , Wada Masamitsu
Published: April 2021

The preprophase band (PPB) of microtubules (MTs), which appears at the future site of cytokinesis prior to cell division in higher plant cells, disappears by metaphase. Recent studies have shown that displacement of the endoplasm from the PPB region by centrifugation delays the disappearance of the PPB. To study the role of the endoplasm in the cell cycle-specific disruption of the PPB, the filamentous protonemal cells of the fern Adiantum capiUus-veneris L. were centrifuged twice so that the first centrifugation displaced the endoplasm from the site of the PPB and the second returned it to its original location. The endoplasm, including the nucleus of various stages of mitosis, could be returned by the second centrifugation to the original region of the PPB, which persists during mitosis in the centrifuged cells. When endoplasm with a prophase nucleus was returned to its original location, the PPB was not disrupted. When endoplasm with a prometaphase-telophase nucleus was similarly returned, the PPB was disrupted within 10 min of termination of centrifugation. In protonemal cells of Adiantum, a second PPB is often formed near the displaced nucleus after the first centrifugation. In cells in which the endoplasm was considered to have been returned to its original location at the prophase/prometaphase transition, the second PPB did not disappear even though the initial PPB was disrupted by the endoplasm. These results suggest that cell cycle-specific disruption of the PPB is regulated by some factor(s) in the endoplasm, which appears at prometaphase, i.e. the stage at which the PPB is disrupted in non-centrifuged cells.
Subcellular imaging of calcium exchange in cultured cells with ion microscopy
Authors: Chandra Subhash , Ausserer Walter , Morrison George H.
Published: April 2021

Calcium to calcium exchange between intracellular and extracellular pools has been imaged directly in individual cells using stable 44Ca (98.78% enrichment) in the nutrient medium and an isotopic imaging technique, ion microscopy. Observations were made by imaging mass 40 to determine the native intracellular calcium (40Ca), and mass 44 to localize the 44Ca from the extracellular medium that exchanged with cellular calcium. LLC-PK, porcine kidney epithelial cells were exposed to the nutrient medium with 1.87 mM stable 44Ca for 0,1, 2, 5, 8, 20, 60 and 90 min, and cryogenically prepared prior to ion microscopic analysis. The cell nucleus, the Golgi region and the remaining cell cytoplasm could be spatially resolved to within about 0.5 fan using the ion microscope. On the basis of the kinetics of 40Ca to 44Ca exchange it was observed that all three compartments had a rapidly exchanging pool of calcium, which took about 2 min to exchange. A moderately rapidly exchanging pool of calcium was identified between 2 and 20 min of calcium exchange. The cells had exchanged about 50% of their total internal calcium with the external calcium in less than 20 min. The remaining 50% of the cellular calcium could be classified as a slowly exchanging pool. Isotopic images of 39K and 23Na were recorded along with 40Ca and 44Ca images to assess the health status of the cell. Isotopic imaging has the unique ability to distinguish intracellular calcium from the extracellular calcium that enters cells and has enormous potential for studies of calcium transport under physiological and pathological conditions.
Differential expression of the VLA family of integrins along the crypt-villus axis in the human small intestine
Authors: Beaulieu Jean-François
Published: April 2021

Regulation of epithelial cell proliferation, migration and differentiation under physiological conditions remains poorly understood. Interaction of the cells with their underlying basement membrane through integrins, a specific subset of cell surface binding proteins, is one potential mechanism. In the present work, I examined this hypothesis by investigating the distribution of a variety of epithelial basement membrane proteins and the expression of the members of the VLA family of integrins in the adult intestinal epithelium. Indeed, this rapidly renewing simple epithelium contains within its functional unit, the crypt-villus axis, essentially two distinct cell populations: the proliferative and undifferentiated crypt cells and the mature enterocytes on the villus. Although immunolocalization of basement membrane molecules revealed that laminin, type IV collagen and heparan sulfate proteoglycan are distributed homogeneously all along the crypt-villus axis, other nonexclusive basement membrane components were found differentially expressed. Tenascin was concentrated at the base of both villus and lower crypt cells while cellular fibronectin was mostly detected in association with the crypt cells. Moreover, VLA β;1 as well as 5 of the 6 VLA α subunits tested were expressed by intestinal epithelial cells under specific patterns of staining. The β1 and α6subunits were strongly detected at the base of all enterocytes while β5 also detected all along the cryptvillus axis, was weaker and consistently appeared with a punctated/interrupted pattern. On the other hand, the VLA α1 α2 and α3 were expressed at the basolateral domains of enterocytes under distinctive crypt-villus gradients. The α1 subunit was detected at the base of all epithelial cells but lateral staining was only observed in differentiating cells (middle and upper crypt). Finally, in most specimens, α2 and α3 displayed strictly complementary staining patterns for the lower crypt region (α2β3—) and the upper crypt-to-villus region (β2 —, β3). Taken together, these data emphasize that proliferation, migration and differentiation in the normal state are susceptible to various influences including compositional changes in the basement membrane and differential expression of receptors for these components.
Collagen-induced rapid morphogenesis of human mammary epithelial cells: The role of the α
2
β
1 integrin
Authors: Berdichevsky Feodor , Gilbert Chris , Shearer Moira , Taylor-Papadimitriou Joyce
Published: April 2021

The cell line MTSVl-7, originally derived by immortalizing mammary epithelial cells cultured from human milk was able to form three-dimensional structures in collagen gel. We have now found that these cells, cultured as a monolayer, are able to undergo rapid morphogenesis forming ridges and balls around collagen fibres, when soluble collagen type I is added to the medium. Monoclonal antibodies to the α2(PlE6) and β1(mAB13) subunits of VLA-2, but not to the α3-subunit (P1B5) of VLA-3, could block this collagen-induced rapid morphogenesis (CIRM). The effect of the antibodies on cell attachment, spreading, and migration on collagen gels was analyzed to identify α2β1 dependent steps which might be involved in CIRM. The results suggest that while other proteins, besides α2α1, are also involved in cell attachment and migration, cell spreading was specifically blocked by antibodies to the VLA-2, but not to the VLA-3 integrin. The results demonstrate that the α2α1 integrin plays a crucial role in the collagen-induced morphogenesis of human mammary epithelial cells and implicate the process of VLA-2-dependent cell spreading as an important step in this morphogenesis.
Nucleolus behaviour during the cell cycle of a primitive dinoflagellate eukaryote,Prorocentrum micansEhr., seen by light microscopy and electron microscopy
Authors: Soyer-Gobillard Marie-Odile , Geraud Marie-Line
Published: April 2021

Light-microscopy observation of the dinoflagellate Prorocentrum micans after silver-staining of the argyrophilic proteins of the nucleolar organizing region (Ag-NOR staining) showed the presence of nucleolar material throughout the vegetative cell cycle, and in particular during all the mitotic stages. This contrasts with the case in most higher eukaryotes, in which nucleoli disappear at the end of prophase and are reconstituted in daughter cells during telophase.

Electron-microscope (EM) observations after conventional or fast-freeze fixation revealed that during interphase several functional nucleoli with three compartments (NORs, the fibrillogranular and the preribosomal granular compartments) are present in a nucleus in which the envelope is persistent and the chromosomes are always compact. During early prophase, when chromosomes are beginning to split, the nucleoli remain functional, whereas in late prophase they contain only a NOR and the granular component, and the chromosomes are surrounded by many protein masses. In early telophase, the nucleolar material coating the chromosomes migrates along with the chromosomes. Nucleologenesis occurs through the formation of prenucleolar bodies around lateral or telomeric nucleofilaments extruding from the chromosomes. Several chromosomes can contribute to the formation of one nucleolus. The behaviour of these ‘persistent nucleoli’ in a closed-nucleus model such as that of the dinoflagellates is discussed with regard to the higher eukaryotes.
Expression and function of ras proto-oncogene proteins in human sperm cells
Authors: Naz Rajesh .K , Ahmad Khaliq , Kaplan Paul
Published: April 2021

The presence and role of c-ras proteins were investigated in mature human sperm cells. The v-H-ras monoclonal antibody (mAh) against the c-ras protein, p21, reacted specifically with the acrosomal region of methanol-fixed as well as unfixed-live capacitated and non-capacitated human sperm cell in the indirect immunofluorescence technique. The v-H-ras mAb predominantly recognized c-ras protein of 21 kDa on the Western blot of lithium diiodosalicylate (LlS)-solubilized human sperm preparation. The incubation of sperm cells with v-H-ras mAb affected the sperm cell function in the human sperm penetration assay. The antibody significantly reduced the acrosome reaction and release of acrosin activity from the sperm cells. There was no effect of the mAb on percentage motility, although the mAb significantly affected various motility characteristics such as linearity, amplitude of lateral head displacement (ALH) and beat frequency, the motility parameters involved in the hyperactivation phenomenon of sperm cells leading to capacitation and acrosome reaction. These results suggest that the c-ras or c-ras-like proteins are present in mature sperm cell and may have a role in capacitation and/or acrosome reaction of human sperm cell.
Effect of caffeine on intracellular transport of Semliki Forest virus membrane glycoproteins
Authors: Kuismanen Esa , Jäntti Jussi , Mäkiranta Virpi , Sariola Merja
Published: April 2021

The effect of caffeine on the intracellular transport of Semliki Forest virus (SFV) membrane glycoproteins was studied in baby hamster kidney (BHK) cells. The movement of the proteins was affected at two steps in the exocytic pathway. The exit of the proteins from the endoplasmic reticulum (ER) was inhibited by 10 mM caffeine at 20°C, a temperature that normally allows transport to the Golgi complex. At higher temperatures (28°C and 37°C) in the presence of 10 mM caffeine exit from the ER occurred, but the proteins accumulated at intracellular membrane elements. Immunofluorescence localization, endoglycosidase-H analysis, and analysis of the proteolytical cleavage of the p62 precursor protein suggested that transport in the presence of 10 mM caffeine was arrested at the membranes between the trans-Golgi and the plasma membrane.
In situ localisation of single-stranded DNA breaks in nuclei of a subpopulation of cells within regenerating skeletal muscle of the dystrophic mdx mouse
Authors: Coulton G. R. , Rogers B. , Strutt P. , Skynner M. J. , Watt D. J.
Published: April 2021

Degeneration of muscle fibres during the early stages of Duchenne Muscular Dystrophy (DMD) is accompanied by muscle fibre regeneration where cell division and myoblast fusion to form multinucleate myotubes within the lesions appear to recapitulate the events of normal muscle development. The mechanisms that govern the expression of genes regulating differentiation of myoblasts in regenerating skeletal muscle are of great interest for the development of future therapies designed to stimulate muscle regeneration. We show here that single-stranded breaks in DNA are localised in nuclei, using an exogenously applied medium containing labelled deoxynucleotides and the Klenow fragment of DNA polymerase I. The nuclei of a sub-population of cells lying in the inflammatory infiltrate of lesions in the skeletal muscle of the muscular dystrophic mouse (mdx), a genetic homologue of DMD, were labelled in this fashion. By contrast, labelled cells were completely absent from the muscles of normal non-myopathic animals (C57BL/10) and non-lesioned areas of mdx muscles. Cells expressing the muscle-specific regulatory gene, myogenin, were also found within mononucleate cells and myotubes within similar mdx muscle lesions.

While we cannot yet say that the cells labelled by the DNA polymerase reaction are in fact differentiating, they were found only in significant numbers within mdx muscle lesions where new muscle fibres appear, providing strong circumstantial evidence that they are intimately associated with the regenerative process. Using a range of nucleases and different DNA polymerases, we show that the DNA polymerase-labelling reaction observed was DNA-dependant and most probably due to infilling of naturally occurring single-stranded gaps in DNA.

Since the regenerative process in human Duchenne Muscular Dystrophy is apparently less effective than that seen in mdx mice, continued study of singlestranded DNA breaks may help to elucidate further the mechanisms controlling the expression of genes that characterise the myogenic process during skeletal muscle regeneration. Such findings might be applied in the development of future therapies designed to stimulate muscle regeneration in human dystrophies.
Isoprenylation of rab proteins on structurally distinct cysteine motifs
Authors: Peter Matthias , Chavrier Philippe , Nigg Erich A. , Zerial Marino
Published: April 2021

rab proteins are low molecular weight GTP-binding proteins highly related to Yptlp and Sec4p, which are involved in the control of secretion in yeast Saccharo-myces cerevisiae. Morphological and biochemical studies have shown that rab proteins are membrane associated and are localized to specific subcompartments along the exocytic and endocytic pathway. Membrane association requires the presence of C-terminal cysteine residues. The present report indicates that the structurally distinct cysteine motifs of rab proteins are subjected to isoprenylation both in vitro and in vivo. Studies on deletion mutants suggest that an intact C-terminal end is required for the association of rab proteins with the membrane and is necessary for the post-translational modification. Finally, we show that the isoprenoid transferase which modifies rab termini is different from the enzyme which farnesylates nuclear lamins and ras proteins in vitro.
Immunolocalization of kinesin in sea urchin coelomocytes: Association of kinesin with intracellular organelles
Authors: Henson John H. , Nesbitt David , Wright Brent D. , Scholey Jonathan M.
Published: April 2021

We have recently used domain-specific monoclonal antibodies (mAbs) to immunofluorescently localize kinesin to vesicle-like structures in the cytoplasm of sea urchin coelomocytes. In order to characterize further these localization patterns we have examined the distribution of kinesin with respect to the arrangement of microtubules (MTs) and various organelles. In double-label experiments involving the immunofluorescent staining of kinesin (using a mixture of the mAbs SUK2, 4 and 5), MTs were labeled with an antiserum against sea urchin tubulin, the endoplasmic reticulum (ER) was labeled with an antiserum against a luminal calsequestrin-like protein, the Golgi apparatus was labeled with rhodamine-wheat germ agglutinin (WGA) or NBD-ceramide, mitochondria were labeled with rhodamine 123, endosomes were labeled with Texas Red-ovalbumin, and lysosomes were labeled with Lucifer yellow or acridine orange. Kinesin-labeled vesicle-like structures were found in the same regions of the cells as MTs and the ER, being widely distributed in motile cells, but restricted to the perinuclear regions of stationary cells. There also appeared to be a correlation between the distribution of endosomes and kinesin staining in a sub-population of cells. The kinesin binding structures were found occasionally to align in linear arrays, consistent with the idea that kinesin may transport ER and endosomes along linear MT tracks. No clear correlations were observed between the kinesin staining and the distribution of mitochondria, the Golgi apparatus or lysosomes, suggesting that kinesin may specifically associate with only a subclass of organelles in coelomocytes.