bluuebunny/crossref_metadata_2025 huggingface dataset:
Magnetic resonance imaging approaching microscopic scale: Maturation stages of Acetabularia mediterranea reproductive caps
Authors: Harrison Lionel G. , Luck S. D. , Munasinghe B. D. J. P. , Hall Laurance D.
Published: April 2021

The reproductive cap of the giant single-celled alga Acetabularia mediterránea (or A. acetabulum) has rays tapering from a width of about 400 μm at the circumference of the cap to about 30μm at their junction with the stalk of the cell. This is ideal geometry for testing the current limits of spatial resolution of proton magnetic resonance imaging. In this work, resolution of features down to 40 μm is achieved. Maturation of the cap rays involves a major cytoplasmic reorganization, from continuous cytoplasm and a central vacuole in each ray to bundles of cysts surrounded by aqueous solution. This work shows that an intermediate stage in the change can be highlighted in images by relaxation time (T1) contrasting.
Relationship between the timing of dna replication and the developmental competence in Acanthamoeba castellanii
Authors: Jantzen Helga , Schulze Ingrid , Stöhr Michael
Published: April 2021

In Acanthamoeba, two different cell types are known. Trophozoites are generated in the mitotic division cycle, whereas cells committed at late G2 phase of the cell cycle develop into cysts in response to starvation. In this paper we study the role of timing of DNA replication in regulating development. The investigation was performed with cultures growing in a non-defined medium (ND cells) that show a high encystation competence and with cultures that have been growing in a chemically defined medium (D cells) for several years and show a low encystation competence. Bivariate DNA/BrdUrd distributions show that ND cells progress through a cycle in which the short replication phase occurs immediately and exclusively after prior completion of mitosis. These cells arrest at late G2 phase of the cell cycle during the stationary stage. In D cells, DNA replication and mitosis seem to be uncoupled, since replication takes place before as well as after mitosis. These cells arrest within their replication phase during the stationary stage. These findings indicate that D cells do not progress into late G2 phase of the cell cycle and hence do not have the competence for commitment. The alternate timing of DNA replication and the low encystation competence of D cells can be reversed by cultivation of these cells in ND medium. Synchronization experiments reveal that late G2 phase ND cells exhibit a low capacity for BrdUrd incorporation and growth after transfer into D medium, whereas ND cells of earlier phases of the cell cycle show premitotic incorporation of BrdUrd into nuclear DNA and growth. These findings suggest on the one hand that premitotic DNA synthesis is a prerequisite for growth of cells in D medium, and that there is a dependence of the induction of premitotic DNA synthesis on the cell cycle, and on the other hand that a reciprocal relationship exists between the capacity of premitotic DNA synthesis and commitment to differentiation.
Three-dimensional organization of chromosomes of Crepis capillaris by optical tomography
Authors: Rawlins D. J. , Shaw P. J.
Published: April 2021

The three-dimensional (3D) organization of chromosomes of Crépis capillaris (2n = 6) has been investigated. Root tips were fixed, macerated with enzymes and gently separated without squashing. The cells were then stained with DAPI and optically sectioned under computer control. Sections were stored as video images and processed to remove noise and out-of-focus information. Computer modelling was then used to trace the paths of each chromosome and to display the paths as a 3D wire diagram. In all, 88 sets of anaphase chromosomes were modelled from 47 optically sectioned cells. The models and the coordinates of the chromosomes were then analysed to detect non-random arrangements or preferential associations of particular pairs of chromosomes. The methods used have significant advantages over electron microscope tomography for the analysis of 3D chromosome arrangement; in particular, the large number of samples allowed more thorough statistical tests to be performed on the data obtained. No evidence was found for either non-random arrangements or homologous association and, moreover, the distances between the two larger pairs of homologues were larger than for other pairs of chromosomes. These results conflict with previous results for this and other plant species where the material was squashed before measurements were taken. We found no evidence of haploid genome separation.
Dissociation of lens fibre gap junctions releases MP70
Authors: Kistler Joerg , Bullivant Stanley
Published: April 2021

MIP and MP70 are putative gap junction components in the plasma membranes of the mammalian lens fibre cells. We show now that MP70 can be solubilized separately from MIP in mild detergent solutions, and that this treatment results in the dissociation of the fibre gap junctions. Solubilized MP70 was isolated as 16’9 S particles by velocity gradient centrifugation and in the electron micro scope had the appearance of short double-membrane structures consistent with connexon-pairs. These observations open a new experimental avenue in which to characterize separately the two putative lens gap junction proteins structurally and functionally.
Parallel development of cadmium resistance and in vitro transformation in cultured indian muntjac cells
Authors: Bouffler S. D. , Ord M. J.
Published: April 2021

The development of cadmium resistance in an Indian muntjac cell line has been investigated. The parent cell line is highly sensitive to cadmium ions. Resistance was obtained by continuous growth of cells in low levels of cadmium with stepwise increments. Four cell lines were developed with resistances of between 50- and 200-fold greater than that of the parental line. Early in the development of resistance an unstable cell line displaying extensive chromosomal rearrangement and an elevated sister chromatid exchange frequency was identified. The more stable resistant lines produced from this original cell line have normal karyotypes. Having passed through the initial period of genome rearrangement the resultant cells acquired several characteristics of morphologically transformed cells. It is concluded that long-term exposure to low levels of cadmium can transform cells in vitro concurrently with their acquiring cadmium resistance.
Ultraviolet microbeam irradiation of microtubules in vitro the action spectrum for local depolymerization of marginal band microtubules in vitro matches that for reducing birefringence of chromosomal spindle fibres in vivo
Authors: Hughes Kimberley , Forer Arthur , Wilson Paula , Leggiadro Cynthia
Published: April 2021

Marginal bands were isolated from newt red blood cells and, using monochromatic light from an ultraviolet microbeam, the marginal band microtubules were irradiated in vitro to produce areas of reduced birefringence (ARBs). The ARBs neither moved nor changed shape after they were formed, though the marginal bands sometimes changed shape during the irradiation. Marginal band ARBs were regions in which the microtubules were locally depolymerized, as determined by electron microscopy and immunofluorescence. The action spectrum for producing ARBs on marginal band microtubules in vitro matches very closely the action spectrum for producing ARBs on crane-fly spermatocyte chromosomal spindle fibres in vivo, which indicates that ARBs in vivo are produced by the ultraviolet light acting directly on the microtubules (as opposed to an intermediate component), and confirms, without complications inherent in the fixation of living cells, that ARBs on spindle fibres in vivo are regions in which microtubules are locally depolymerized.
Analysis of a Dictyostelium chemotaxis mutant with altered chemoattractant binding
Authors: Segall Jeffrey E. , Bominaar Anthony A. , Wallraff Eva , J. W. De Wit René
Published: April 2021

A Dictyostelium discoideutn mutant defective in folate chemotaxis has been analysed using biochemical, behavioural, and genetic methods. A subset of the cell-surface folate binding sites appeared to be locked in a high-affinity state from which folate dissociated extremely slowly. Changes in cell area and motility induced by step increases in folate required 10-to 100-fold higher concentrations than in the wild type. Folate-stimulated cyclic GMP production was also altered. Chemotactic responses to cyclic AMP as well as cyclic AMP-stimulated cyclic GMP production were normal. The mutation responsible for the chemotaxis defect, termed folA1OOO, was localized to linkage group IV. The alterations in folate binding and sensitivity to folate co-localized with the folA1OOO mutation. We conclude that the folA1OOO mutation arrests the folate chemotaxis receptor in a high affinity state that can only poorly transduce folate binding into chemotactic responses.
Distinct controls of dna replication and of nuclear division in the cell cycles of the chlorococcal alga Scenedesmus quadricauda
Authors: Zachleder V. , Šetlík I.
Published: April 2021

In the course of the cell cycle of Scenedesmus quadricauda, the syntheses of RNA and total protein occur in steps. Each step represents an approximate doubling of the preceding amount of RNA or protein per cell. The increase in protein content per cell runs parallel to, but with a constant delay behind, the corresponding RNA steps. When protein synthesis is suppressed (e.g. by maintaining the cells in the dark) after an RNA synthesis step has already occurred the cells double their DNA content, but no corresponding nuclear division occurs and uninuclear daughter cells with double the amount of DNA may be formed. Under conditions of phosphorus or nitrogen starvation RNA synthesis is stopped while protein synthesis continues. In this case, the number of DNA replication rounds corresponds to the reduced RNA content while the number of nuclear divisions tends to follow the number of protein synthesis steps until one genome per nucleus is attained. These results indicate that with each doubling of RNA content the cells become committed to DNA replication, while doubling of protein content is required for the commitment to the corresponding nuclear divisions.
Serum from partially hepatectomized rats induces primary hepatocytes to enter s phase: A role for prostaglandins?
Authors: Little Penelope , Skouteris George G. , Ord Margery G. , Stocken Lloyd A.
Published: April 2021

Serum obtained from partially hepatectomized rats 1, 3 or 24 h after operation was more effective in stimulating DNA synthesis in primary adult rat hepatocytes than serum from sham-operated rats; exposure to the serum for 2h was sufficient to promote growth. Serum from the partially hepatectomized rats contained elevated levels of PGE2 and PGF2α; it promoted hepatocytes to release prostaglandins into their culture medium. Growthpromoting effects of the serum and its capacity to elicit prostaglandin release into the culture medium were inhibited by 0*1 mM-indomethacin or 1 mM-aspirin. 0–1 mM-indomethacin also prevented DNA synthesis if the inhibitor were added 4h after growth had been initiated by serum from partially hepatectomized rats, suggesting that prostaglandins continue to be important for the maintenance of hepatocyte growth for at least 6h.
Neuronal maintenance and neurite extension of adult mouse neurones in non-neuronal cell-reduced cultures is dependent on substratum coating
Authors: Orr Douglas J. , Smith Robert A.
Published: April 2021

Adult mouse DRG neurones have been maintained for 14 days in cultures where non-neuronal cell proliferation was inhibited by the inclusion of 5xlO~6/iM-cytosine arabinoside (AraC) in the medium from the onset of culture. On uncoated plastic neurone numbers significantly declined in the absence of non-neuronal cell outgrowth compared with uninhibited co-cultures. However, when neurones were maintained in the presence of AraC on certain coated surfaces this decrease in neurone numbers was not observed. Combinations of fibronectin (FN) and laminin (LAM) proved most effective for 7 and 14 days in vitro, although either was beneficial if used separately. Microexudates produced by the fibroblast line, 3T6, also significantly improved neuronal counts for 14 days in vitro. However, a microexudate derived from primary cultures of mouse hepatocytes, although advantageous for 7 days in vitro, was not effective in maintaining neurones over the 14-day culture period, reminiscent of previous observations when synthetic cationic agents were used. Electrophoretic analysis of the fibroblast exudate indicated that fibronectin was present in the substrate-attached material generated by this cell line.

The reduction in non-neuronal cell growth facilitated the monitoring of neuronal structural detail by scanning electron microscopy. Examination of neurite extension, indicative of neurone differentiation, was particularly improved. FN/LAM and the fibroblast-derived exudate increased nerve fibre growth, whilst the hepatocyte exudate had little effect on neurite regeneration, and polylysine had a detrimental effect.

The data demonstrate that substrata can have a significant effect on maintenance and differentiation of adult neurones in primary culture. The use of cultured adult sensory neurones instead of only foetal ones in neurobiological research is therefore now a plausible alternative.
Cloning and sequencing of the cyclin-related Cdc13
+ gene and a cytological study of its role in fission yeast mitosis
Authors: Hagan Iain , Hayles Jacqueline , Nurse Paul
Published: April 2021

We have cloned and sequenced the cdcl3+gene from fission yeast. When a major part of the cdc13+ gene is deleted from the chromosome, cells arrest in interphase, but partial loss of gene activity leads to cells containing condensed chromosomes, aberrant septa and a microtubular cytoskeleton with characteristics of both G2 and M. Expression of this phenotype is influenced by the nutritional status of the cell. Our results suggest that the cdc13+gene function is required for the control of the G2 to M transition. It appears to play a role in regulating the separate pathways of events involved in the physical process of mitosis, for example in the reorganization of the cytoskeleton on transition from G2 to mitosis. The cdc134- gene function interacts closely with both the yeast and human homologues of cdc2+ suggesting that mammalian cells may contain a cdc13+ homologue. The gene encodes a putative polypeptide of 482 amino acids, and a central region of 176 amino acids of this polypeptide is 50% identical with sea urchin cyclin. Therefore, the cdc13+ protein is cyclin related and could act as a regulator or substrate of the p34cdc2 protein kinase, which initiates mitosis.
Remodeling of mouse thymocyte nuclei depends on the time of their transfer into activated, homologous oocytes
Authors: Szöllösi Daniel , Łowska Renata , Szöllösi Maria S. , Tarkowski Andrzej K.
Published: April 2021

The potential of parthenogenetically activated mouse oocytes to remodel somatic cell nuclei was studied by ultrastructural means using oocytethymocyte hybrids. Complete nuclear remodeling, initiated by nuclear envelope breakdown and chromosome condensation (which is followed by formation of pronucleus-like nucleus) is possible only during a short time gap between metaphase II and telophase of meiotic division. Maturationpromoting factor activity is high during this period. The thymocyte nucleus can follow the sequence of morphological changes only in concert with the development of the native nucleus and only after exposure of the chromatin to the ooplasm. If hybridization is effected with pronucleate oocytes, the thymocyte nucleus retains its interphase character but shows particular modifications in nucleolar morphology (identical to changes observed during reactivation of the nucleolus in stimulated lymphocyte) and in the activity of the nuclear envelope (blebbing). Thus the nucleus not exposed to maturation-promoting factor activity may be influenced by a ‘programme’ specific for oocyte (blebbing) and by a programme inherent in the introduced somatic cell nucleus.
Cytoplasmic to nuclear translocation of RNA polymerase I is required for lipopolysaccharide-induced nucleolar RNA synthesis and subsequent DNA synthesis in murine B lymphocytes
Authors: Halleck Margaret S. , Schlegel Robert A. , Rose Kathleen M.
Published: April 2021

The synthesis of ribosomal RNA (rRNA) in murine B lymphocytes is markedly elevated in response to mitogens such as lipopolysaccharide (LPS). First, to investigate the mechanism involved, antibodies directed against RNA polymerase I, the enzyme responsible for transcription of ribosomal genes, were introduced into the cytoplasm of lymphocytes via red cell-mediated microinjection and the ability of cells to synthesize RNA was examined. Simultaneous immunofluorescence/autoradiography revealed that 7% or less of the cells injected with specific antibodies prior to stimulation were actively synthesizing rRNA 15 or 40 h following LPS addition. In contrast 19 % and 27 % of cells injected with control IgG were active at these times. Non-ribosomal RNA synthesis was unaffected by the presence of anti-RNA polymerase I antibodies. Since antibodies injected into the cytoplasm were limited to that compartment, these data suggest that rRNA synthesis induced by LPS requires translocation of cytoplasmic RNA polymerase I into the nucleus. Second, to test whether synthesis of rRNA is required for entry into S phase, the effect of anti-RNA polymerase I antibodies on DNA synthesis in response to LPS was evaluated. Only 7 % of cells containing anti-RNA polymerase I antibodies had initiated DNA synthesis 40 h after LPS addition whereas 25 % of cells containing control IgG were actively synthesizing DNA at that time. These results suggest that nuclear accumulation of RNA polymerase I and increased rRNA synthesis are required for LPS-induced DNA synthesis in B lymphocytes.
Characterization of the fission yeast cdc10
+ protein that is required for commitment to the cell cycle
Authors: Simanis Viesturs , Nurse Paul
Published: April 2021

We have used antiserum raised against a β-galacto-sidase-cdcld4- fusion protein to identify the protein product of the cdcHT start gene of Schizosaccharo-myces pombe. This gene is required for progress through the Gi phase of the cell cycle and for activating processes such as the increase in histone mRNA level in preparation for S phase. The protein has an apparent molecular weight of 87 000 and is phosphorylated on multiple serine residues. The protein remains phosphorylated throughout the mitotic cell cycle and shows no significant steady-state changes in level. The antiserum has also detected a protein similar in size to pSTcdc10 in human cells.
Microtubules are acetylated in domains that turn over slowly
Authors: Webster Daniel R. , Borisy Gary G.
Published: April 2021

Tubulin is subject to a post-translational acetylation reaction that is thought to be correlated with increased stability of the modified microtubules (MTs). We sought to test directly the stability of acetylated MTs by determining their specific rate of turnover. We used human fibroblasts, which contain a subset of MTs that display terminal and internal domains of acetylation. The turnover of acetylated domains was analysed by microinjecting cells with biotinylated brain tubulin and determining, by triple-label immunofluorescence, the progress of incorporation of biotinylated tubulin into acetylated and non-acetylated domains. Within two minutes after injection, biotinylated domains were contiguous with virtually all observed non-acetylated MT ends but were not contiguous with terminal acetylated domains, demonstrating that the former were growing while the latter were not. Ten minutes after injection, many MTs lacking acetylated domains had incorporated biotinylated subunits uniformly while most MTs containing acetylated domains remained unlabelled, indicating that non-acetylated MTs were turning over while most acetylated domains were not. One hour after injection, virtually all non-acetylated MTs were labelled with biotin whereas approximately half of the acetylated domains contained biotin, demonstrating that acetylated domains turned over much more slowly than the non-acetylated, bulk array. Nonacetylated MT regions flanking acetylated domains also lacked hapten, indicating that acetylation modified discrete regions along stable MTs. Sixteen hours after injection, cells that had not entered mitosis still retained acetylated domains that had not turned over (13% of all acetylated domains), indicating that acetylated domains can be extremely long-lived. Prophase cells displayed no acetylated tubulin staining, indicating that the maximum lifetime of cytoplasmic acetylated domains was one cell cycle. The results show that in this cell line only limited domains of stable MTs usually become acetylated, and that the turnover of these domains and the MTs that bear them is much slower than that of the bulk array.
Cells: Principles of Molecular Structure and Function
Authors: Downes C S.
Published: April 2021

This textbook is intended to provide a manageable first course in cell biology, for students whose previous acquaintance with the subject and with several underlying disciplines may be rather slight. It succeeds in this aim, and is therefore a valuable addition to the range of texts available, shorter and conciser than the alternatives. There has been no equivalent introductory text since R. D.Dyson’s ‘Cell Biology: a Molecular Approach’, a useful contribution in its time, but now ten years out of date.
Functional recombination of outer dynein arms with outer arm-missing flagellar axonemes of a Chlamydomonas mutant
Authors: Sakakibara Hitoshi , Kamiya Ritsu
Published: April 2021

A flagellar mutant of Chlamydomonas, oda, lacks the entire outer dynein arm but can swim at a speed of one third to half of that of the wild type. We found that the addition of a high-salt extract of wild-type axonemes to demembranated oda cell models restored up to 83 % of the outer arms normally present on the outer-doublet microtubules of wild-type axonemes. Furthermore, when reactivated in the presence of ATP after being mixed with the extract, the oda cell models gained a higher level of motility, close to that of the wild type. The increase in flagellar beat frequency parallelled the increase in the number of restored outer dynein arms. These observations indicate that the axoneme of the oda mutant retains the binding sites for the outer dynein arms, and that the outer arms solubilized with high salt are functionally active. This in vitro recombination system with the oda mutant should be useful as an assay system for various preparations of outer-arm dynein. Evidence is presented that the two axonemes on an oda cell model beat at the same frequency, whereas those on a wild-type model beat at different frequencies. The two oda axonemes beat at the same frequency even after the higher level of motility has been restored by addition of crude dynein extract. We propose that a heterogeneity in the outer dynein arms is responsible for the frequency imbalance between the two flagella of wild-type Chlamydomonas.
Differential partitioning of plasma membrane proteins into the Triton X-100-insoluble cytoskeleton fraction during concanavalin A-induced receptor redistribution
Authors: Patton Wayne F. , Dhanak Michelle R. , Jacobson Bruce S.
Published: April 2021

The plasma membrane proteins of Dictyostelium discoideum were characterized with respect to their partitioning into the Triton-insoluble cytoskeleton fraction of the cell during concanavalin A-induced capping. Two fractions of plasma membrane-associated concanavalin A were identified; one that immediately associated with the cytoskeleton fraction via cell surface glycoproteins, and one that partitioned with the cytoskeleton only after extensive cell surface glycoprotein cross-linking. Three major classes of polypeptides were found in the plasma membrane that differed with respect to their partitioning properties into the cytoskeleton fraction. The temporal order of association of the polypeptides with the cytoskeleton during concanavalin A-induced capping corresponded to the strength of their association with the cytoskeleton fraction as determined by pH and ionic strength elution from unligated cytoskeletons.
Mitosis in Drosophila
Authors: Glover David M.
Published: April 2021

Drosophila is an attractive organism in which to study both the rapid rounds of mitosis typical of embryonic development in many species, and the longer cell cycles of diploid tissues later in development. Mutations in genes essential for mitosis in Drosophila may result in lethality in late embryonic, larval or pupal stages of development. In addition, mutations in many genes required for the nuclear divisions of early embryogenesis have been found in screens for female sterility. The mitotic mutations have phenotypes indicative of lesions at a variety of mitotic stages. A combined molecular and genetic analysis of these genes has the potential to unravel the complex set of protein-protein interactions that occur in this dynamic process.
A gene for the suppression of anchorage independence is located in rat chromosome 5 bands q22-23, and the rat alpha-interferon locus maps at the same region
Authors: Islam M. Quamrul , Szpirer Josiane , Szpirer Claude , Islam Khaleda , Dasnoy Jean-Francois , Levan Göran
Published: April 2021

Cell hybrids between malignant mouse hepatoma cells and normal rat fibroblasts with approximately one set of chromosomes from each parent exhibited remarkable karyotypic stability. Most chromosomes of both parents were retained even after prolonged culture in vitro. Normally, such hybrids showed suppression of the transformed phenotype and formed no colonies in soft agar. However, two hybrids, BS140 and BS181, formed a few colonies in soft agar when many cells were seeded, and also occasional foci of cells were detected piling up in monolayer cell cultures. We isolated soft agar colonies (a-subclones) and subclones from foci (h-subclones) of both hybrids, and, as a control, subclones of cells from random areas without foci of one hybrid (BS181 p-subclones). When tested for soft agar growth, cells from the a- and h-subclones of both BS140 and BS181 formed colonies at frequencies comparable to the malignant mouse hepatoma parent, whereas the control cells of the BS181 p-subclones (like the normal rat parental cells) yielded no soft agar colonies. All the cell lines were subjected to detailed karyotype analysis in G-banding, which resulted in the finding that cells from the original BS140 hybrid contained at least one copy of each rat chromosome, whereas BS140 a- and h-subclones had lost both copies of rat chromosome 5. Similarly, the original BS181 hybrid contained at least one copy of each rat chromosome, whereas BS181 a- and h-subclones displayed a deletion of the segment q22-23 of rat chromosome 5. In contrast, the control BS181 p-subclones contained one or two copies of non-deleted rat chromosome 5. The conclusion is that a gene for the suppression of anchorage independence is located in the segment 5q22-23. We propose to call this gene SAI1 (for suppression of anchorage independence).

Using Southern blotting, we tested whether any of several gene probes, known to correspond to DNA sequences in rat chromosome 5, were homologous to sequences in the deletion. Only one probe, corresponding to the active alphai-interferon gene, was shown to be located within the deletion. Hence, the SAI1 gene is closely linked to the alpha 1 -interferon gene, and might be identical to this locus.
Potassium depletion inhibits the intracellular transport of secretory proteins between the endoplasmic reticulum and the golgi complex
Authors: Judah Jacob D. , Howell Kathryn E. , Taylor Janet A. , Quinn Paul S.
Published: April 2021

In this paper we show that hepatocytes that have been depleted of K+ secrete albumin, α-1-anti-trypsin and transferrin at a slower rate than cells to which K+ has been returned. K+ depletion has no effect on the intracellular nucleotide pools, and we provide evidence that the inhibitions of secretion caused by depletion of K+ and depletion of ATP are independent.

Studies of the processing of α-1-anti-trypsin show that K+ depletion inhibits the formation of the mature form of the protein, but that immature forms are never secreted. In cells to which K+ was returned, secretion of the mature form was restored. This implies that transport is blocked at a point before the proteins reach the processing enzymes. Proteins delayed by K+ depletion are not removed from the secretory pathway, but are free to mix with protein synthesized subsequently.

These data are supported by subcellular fractionation experiments, which show that the secretory proteins are delayed before reaching the Golgi complex, and by immunoelectron microscopic studies. These show that in K+-deficient cells the morphology of both the endoplasmic reticulum and the Golgi complex is normal. The secretory proteins are trapped in smooth vesicles that contain reaction product when incubated for glucose-6-phosphatase, a marker for the endoplasmic reticulum.
mrnas and a cloned histone gene are differentially expressed during anther and pollen development in rice (Oryza sativa L.)
Authors: Raghavan V.
Published: April 2021

Spatial and temporal changes in the distribution of mRNA sequences during anther and pollen development in rice (Oryza sativa) were investigated by in situ hybridization with [3H] polyuridylic acid ([3H]poly(U)) and a cloned rice histone gene probe. Annealing of sections with [3H]poly(U) showed that poly(A)-containing RNA (poly(A)+RNA) was uniformly distributed in the cells of the anther primordium. During the formation of the archesporial initial, the primary parietal cell, the primary sporogenous cell and tapetum, there was no differential accumulation of poly(A)+RNA in their progenitor cells. Preparatory to meiosis, there was a sharp decrease in poly(A)+RNA concentration in the epidermis and middle layer of the anther wall, although the label persisted in the endothecium, tapetum and microsporocytes. Poly(A)+RNA concentration decreased in these cell types during meiosis and attained very low levels in the disintegrating tapetum and the persistent endothecium of the post-meiotic anther. Pollen development was characterized by the absence of [3H]poly(U) binding sites in the uninucleate microspores and by their presence in the vegetative and generative cells of the bicellular pollen grain. In anther sections hybridized with [3H]histone probe, gene expression was only detected in the endothecium of the premeiotic anther and in the bicellular pollen grains.
Cytoplasmic, nuclear, membrane-bound and secreted [35S]methionine-labelled polypeptide pattern in differentiating fibroblast stem cells in vitro
Authors: Francz P. I. , Bayreuther K. , Rodemann H. P.
Published: April 2021

Methods for the selective enrichment of various subpopulations of the human skin fibroblast cell line HH-8 have been developed. These methods permit the selection of homogeneous populations of the three mitotic fibroblast cell types MF I, II and III, and the four postmitotic cell types PMFIV, V, VI and VII. These seven cell types exhibit differentiation-dependent and cell-type-specific patterns of [35S]methionine-labelled polypeptides in total soluble cytoplasmic and nuclear proteins, also in membrane-bound proteins, and in secreted proteins. In the differentiation sequence MF II - MF III - PMF IV - PMF V - PMF VI 14 cell-type-specific marker proteins have been found in the cytoplasmic and nuclear fraction, also 24 cell-type-specific marker proteins have been found in the membranebound protein fraction, and 11 cell-type-specific marker proteins in the secreted protein fraction. Markers in spontaneously arising and experimentally selected or induced populations of a single fibroblast cell type were found to be identical.
NCL-SG3: A human eccrine sweat gland cell line that retains the capacity for transepithelial ion transport
Authors: Lee Catherine M. , Dessi John
Published: April 2021

An ion-transporting human epithelial cell line, NCL-SG3, has been established by simian virus 40 (SV40) infection of primary cultures from eccrine sweat glands.

The line has been passaged 38 times (over 100 population doublings), has an aneuploid karyotype but has not undergone any ‘crisis’. The cells have retained epithelial morphology and expression of cytokeratin, the intermediate filament characteristic of epithelial cells. Approximately 85% of the population shows at least weak co-expression of vimentin, an intermediate filament associated with mesenchymal and some other non-epithelial cell types in vivo. In addition, SV40 large T-antigen is present, in a predominantly nuclear localization.

Electrically resistant cell sheets are formed on dialysis tubing and cellulose-ester permeable supports. Electrogenic ion transport can be stimulated by the β-adrenergic agonist isoproterenol (10−6M) and by lysylbradykinin (10−7M) but not by the cholinergic agonist carbachol at 10 −6M.
The kinetics of cell-substratum detachment mediated by trypsin: A comparison of normal and duchenne fibroblasts
Authors: Simon L. V. , Pizzey J. A. , Jones G. E.
Published: April 2021

In previous studies of cell-cell and cell-substratum adhesion, we have identified differences in the behaviour between human skin fibroblasts cultured from normal individuals and patients with Duchenne muscular dystrophy (DMD). In these studies, monolayer cultures were dissociated by trypsinization. and no detectable difference was noted in the efficiency of cell dissociation between normal and DMD fibroblast cultures. However, a detailed study by Kent has suggested that Duchenne fibroblasts exhibit increased sensitivity to trypsin. We have re-investigated this finding using an assay that directly measures the number of cells remaining attached to a substratum following trypsinization. In a series of experiments using cultures derived from five normal and five DMD individuals, we can detect no significant difference in the trypsin-induced detachment rates between normal and DMD skin fibroblasts. This observation applies to both growth-phase and stationary-phase cell cultures. This inconsistency with previously reported data on the trypsin-sensitivity of DMD cells is considered in terms of the different assays used and the effects of trypsin on cell-cell and cell-substratum adhesion. The relationship between abnormalities in the behaviour of DMD cells and the localization and primary structure of the DMD gene product are also discussed.
Ultrastructural changes accompany inhibition of proteoglycan synthesis in chondrocytes by cyclofenil diphenol
Authors: Lancaster Carole A. , Fryer Patricia R. , Griffiths Stephen , Mason Roger M.
Published: April 2021

Cyclofenil diphenol, a weak non-steroidal oestrogen, profoundly inhibits [35S] proteoglycan synthesis in cultures of Swarm chondrosarcoma chondrocytes under conditions in which protein synthesis is only marginally reduced. In the present experiments it was shown that after a 40-min treatment with Cyclofenil diphenol (90 μg ml1) most of the normally abundant Golgi stacks in these cells disappeared and after 60 min they were absent. After 2 – 3 h treatment the cisternae of the endoplasmic reticulum (ER) were grossly distended and transformed into large ribosome-studded vesicles containing flocculent and filamentous material. These changes were dependent on the concentration of Cyclofenil and were fully reversible within 21 h of withdrawing the drug.

The ultrastructural changes differed in some aspects if protein synthesis was blocked with cycloheximide for 15 min or 180 min before and during treatment with Cyclofenil. The Golgi disappeared but the ER cisternae, though distended, formed a continuous network and swollen ribosome-studded vesicles did not develop. However, non-membrane-bounded structures containing lipid droplets and material of low electron density developed in the cytoplasm under these conditions.

The ultrastructural changes induced by Cyclofenil differ from those induced by monensin and diethylcarbamazine, suggesting that the drug acts at a different point in the secretory pathway for macromolecules.
Identification of a nucleic acid-regulated cyclic gmp-binding activity in Dictyostelium discoideum
Authors: Parissenti A. M. , Coukell M. B.
Published: April 2021

Using ion-exchange chromatography, we have identified and isolated two forms of a cyclic GMP-specific binding activity in filter-broken cell extracts of Dictyostelium discoideum. Upon addition of excess cold ligand, one form (S-type) released bound 3H-labelled cyclic GMP very slowly (t½ ≈ 68 min), while the other form (F-type) released the cyclic GMP in <1 min. After photoaffinity labelling with 32P-labelled cyclic GMP, both forms revealed a major 160×103Mr band (and a few bands of lower molecular weight) on autoradiograms of sodium dodecyl sulphate-polyacrylamide gels. Addition of 500mM-NaCl to S-type activity converted the activity to a fast-dissociating form indistinguishable from F-type, and this conversion was reversed by dialysis. Salt treatment or dialysis had no appreciable effect on the association/dissociation kinetics of F-type activity. When crude S-type activity was heated (to destroy cyclic GMP binding) and then added to F-type activity, the latter activity acquired slow-dissociating properties identical to S-type. This result suggested that the cells possess a ‘factor’ that can dramatically alter the binding properties of this cyclic GMP-binding protein. Crude preparations of this factor were unaffected by boiling or proteases, but were sensitive to RNase A. Further studies revealed that nucleic acids (in particular, DNA) could effectively mimic the factor in its ability to modulate the binding kinetics of the cyclic GMP-binding activity.
Three-dimensional chromosome arrangement of Crepis capillaris in mitotic prophase and anaphase as studied by confocal scanning laser microscopy
Authors: Oud J. L. , Mans A. , Brakenhoff G. J. , Van Der Voort H. T. M. , Van Spronsen E. A. , Nanninga N.
Published: April 2021

To estimate the extent of ordering of chromosomes, confocal scanning laser microscopy was used to make three-dimensional images from optical sections. For Crepis capillaris, which has 2n = 6 easily recognizable chromosomes, a statistically significant sample of 75 Feulgen-stained root tip anaphases was analysed. A comparison of the observed chromosome ordering and the expected random distribution showed a significant surplus of one of the arrangements with a juxtaposition of the two chromosomes with a nucleolus organizer region. Two of the arrangements with these chromosomes in opposite positions were never observed in our material. Another analysis of 30 mithramycin A-stained prophases and 30 meta- and anaphases showed partly different patterns of non-random chromosome distribution in the two stages of mitosis. A preference for an association of the homologues was observed for all pairs of chromosomes in prophase cells, whereas in meta- and anaphase the association only persisted for the nucleolus organizer chromosomes. This indicates that there may be some relocation of the chromosome positions during the transition from prophase to metaphase. In meta- and anaphase one of the arrangements with juxtaposed NOR chromosomes was preferred, i.e. the ordering in which chromosomes 1 and 3 occupied alternate positions. Probably, the nucleolus is an important factor in producing a non-random distribution, but there could be other factors that influence chromosome ordering as well. A comparison of the anaphase chromosome ordering in C. capillaris plants from very different localities, indicated that the observed non-random distribution was independent of the origin of the material. Existing models of chromosome disposition are not sufficient to explain the observed non-random chromosome ordering in C. capillaris.
Microinterferometry of the movement of dry matter in fibroblasts
Authors: Brown A. F. , Dunn G. A.
Published: April 2021

We describe the use of interferometric microscopy coupled with a novel application of Sénarmont compensation for detecting and quantifying the distribution of dry matter in cultured cells. In conjunction with video techniques and digital image processing, a two-dimensional, calibrated map of the dry mass distribution in an isolated cell can be obtained and digitally recorded. We have called the technique Digitally Recorded Interferometric Microscopy with Analyser Shift (DRIMAS). The method greatly facilitates the automatic recognition of cells by computer. Recorded time-lapse sequences can be used to establish a database of the growth and motility of specific cells in given experimental conditions.

Databases of this type can be analysed to reveal the patterns of growth and locomotory behaviour of individual cells. We describe a systematic method of obtaining parameters of cell size, shape, spreading, intracellular motility and translocation. Autocorrelations and cross-correlations between these parameters can be detected and quantified using time series analysis, revealing potential cause/effect relationships in the mechanisms of growth and motility.

Besides characterizing the overall pattern of cell behaviour, these data can also yield information about the instantaneous pattern of intracellular motility. We describe the use of finite element analysis to reveal the dynamics of the intracellular transport of dry matter. This yields the pattern of the minimum flow of dry matter required to account for the changes in its distribution. Most of this flux is not associated with the movement of visible structures and possibly represents the transport of dissociated components of the cytoskeleton. In chick heart fibroblasts, surprisingly high velocities of nearly 2·0 μm s−1 were detected during the period of increased motility following tail detachment. The total kinetic energy associated with the dry mass flux is a single parameter, which characterizes the instantaneous motility of the cell. We found that the kinetic energy of intracellular motility can be several hundred times greater than the kinetic energy of translocation. Kinetic energy may prove to be a very informative single measure of intracellular motility for assessing the effects of malignant transformation, genetic manipulations, and other experimental treatments on the locomotory machinery of the cell.
Surface views of spermatozoa as revealed by fracture-flip
Authors: Forsman Catarina Andersson , Silva Pedro Pinto Da
Published: April 2021

We have used fracture-flip to produce new, macromolecular-resolution images of the surface of boar spermatozoa. Over the head, acrosomal and postacrosomal regions display sharply demarcated, subtly different surface textures. The rim is particle-poor, as well as a region above the oblique cords over the posterior ring. The tail shows two morphologically distinct domains: (1) the principal piece is covered by a high density of parallel-helical strands and a high density of large globular particles; (2) the midpiece and the neck are covered by smaller particles with apparent random distribution. Rectangular surface specializations frequently seen near the annulus display a waffle-like texture. With the notable exception of the parallelhelical strands of the principal piece the fractureflip images of the boar spermatozoon can be directly related to the freeze-fracture morphology of its plasma membrane.
Quantitative analysis of membrane and secretory protein processing and intracellular transport
Authors: Noe Dennis A. , Delenick Joann C.
Published: April 2021

Quantitative experimental data from studies of intracellular processing and transport of secretory and membrane proteins are only rarely evaluated using kinetic modelling. Instead, the analysis of such data is usually limited to calculating either the apparent half-life or the transit time. Neither of these parameters accurately measures rates of cellular processing or transport. This paper describes a formal approach for the calculation of rate constants, half-lives and lag times for single and sequential processing and transport steps. Generalized kinetic models are presented that can be used to analyse data from both pulse-chase and continuous incubation experiments with labelled amino acids or sugars. The results from a number of published studies are reanalysed using the models to demonstrate the usefulness of kinetic modelling in the routine analysis of experimental data.
Collagenase secretion accompanying changes in cell shape occurs only in the presence of a biologically active cytokine
Authors: Kuter Irene , Johnson-Wintj Barbara , Beaupre Nancy , Gross Jerome
Published: April 2021

We have investigated the relationship between collagenase production, cell shape and stimulatory factors in cell culture. In a homogeneous culture of primary rabbit corneal stromal cells, shape change induced by a variety of agents was not effective in stimulating collagenase secretion. Only in the presence of a biologically active cytokine or phorbol myristate acetate was a correlation seen between changes in cell shape (induced by a second agent) and collagenase secretion by these primary cells. Cell shape changes were not, however, necessary for collagenase secretion, since certain concentrations of endotoxin or lactalbumin hydrolysate effected secretion of the enzyme in the absence of morphological changes. With passaged cells or mixed cell cultures, where cell shape change did correlate with collagenase secretion without the addition of an exogenous agent, the production of an effective cytokine (autocrine or paracrine) was demonstrated. Thus cell shape change seems to be neither universally necessary nor sufficient for the stimulation of collagenase secretion. It is proposed that the function of cytokines may be more immediately related to gene expression in this system than is change in the shape of the cell. The hypothesis is presented that cell shape changes may render the target cells receptive to cytokines, perhaps by replacing the need for a natural cytokine cofactor. It is also demonstrated here that the use of passaged cells, mixed cell cultures containing endogenous cytokine-secreting cells or tissue culture additives can profoundly affect the interpretation of the effect of various agents on collagenase secretion, and may lead to observations that are not directly relevant to cell function in vivo.
Gelatinase (type iv collagenase) immunolocalization in cells and tissues: Use of an antiserum to rabbit bone gelatinase that identifies high and low M
r forms
Authors: Murphy G. , Hembry R. M. , McGarrity A. M. , Reynolds J. J. , Henderson B.
Published: April 2021

An antiserum was raised to rabbit bone gelatinase (type IV collagenase). It was shown by immunoblotting to detect both the low Mr (72000) enzyme produced by connective tissue cells from rabbit, pig, human and mouse, as well as the high Mr (94000–97000) enzymes secreted by macrophages and polymorphonuclear leucocytes from these species, and by rabbit chondrocytes and endothelial cells. Crossed immunoblotting, antibody inhibition and deglycosylation studies indicated that the high and low Afr forms of gelatinase are immunologically distinct gene products, although their substrate specificity profiles are identical.

The anti-gelatinase antiserum was used to immunolocalize the enzyme. Gelatinase was most efficiently detected in rabbit monocytes and connective tissue cells, but cells derived from the human and pig gave poor immunostaining, although mouse gelatinase stained well. The anti-gelatinase antiserum stained cells of the synovial tissue of rabbits at 14 days after induction of an antigen-induced arthritis, demonstrating its usefulness as a tool to assess the role of this enzyme in degradative events.