bluuebunny/crossref_metadata_2025_split huggingface dataset:
A simple formulation of microtubule dynamics: quantitative implications of the dynamic instability of microtubule populations in vivo and in vitro
Authors: Bayley Peter M. , Schilstra Maria J. , Martin Stephen R. 
Published: April 2021

ABSTRACT
A simple formulation of microtubule dynamic instability is presented, which is based on the experimental observations by T. Horio and H. Hotani of coexisting, interconverting growing and shrinking microtubules. Employing only three independent, experimentally determined parameters for a given microtubule end, this treatment accounts quantitatively for the principal features of the observed dynamic behaviour of steady-state tubulin microtubules in vitro. Experimental data are readily reproduced for microtubule length redistribution, and for the kinetics of tubulin exchange processes, including pulse-chase properties. The relative importance of dynamic incorporation and that due to treadmilling are assessed. Dynamic incorporation is found to dominate the overall exchange properties; polarized incorporation due to treadmilling generally becomes significant only when the dynamics are largely suppressed.
This treatment also permits simulation of certain cellular phenomena, showing how microtubule renucleation can control microtubule growth, by means of changes in microtubule number concentration in a system at constant microtubule mass.
A relatively simple extension of the formulation accounts quantitatively for non-steady-state microtubule properties, e.g. dilution-induced rapid disassembly and the oscillatory mode of microtubule assembly. The principles relating dynamic instability and oscillatory behaviour are clearly indicated. Possible mechanisms of the switching of microtubules are briefly discussed.
Differences in intracellular transport of a fluorescent phosphatidylcholine analog in established cell lines
Authors: Sleight Richard G. , Abanto Marina N. 
Published: April 2021

ABSTRACT
The transport and metabolism of a fluorescent phosphatidylcholine analog, l-palmitoyl-2-(N-4-nitrobenzo-2-oxa-l,3-diazole)-aminocaproyl-phos-phatidylcholine ((palmitoyl, C6-NBD)-PC), in BHK, CH0-K1, CHO-15B, MDCK, VA-2, Vero, V79 and WI-38 cells has been investigated. When liposomes containing (palmitoyl, C6-NBD)-PC were incubated with cells at 2°C, spontaneous transfer of the fluorescent lipid from the liposomes to the cells’ plasma membranes occurred. Most of the lipid transferred to the cells could be removed by incubating the cells in the presence of nonfluorescent liposomes or media containing 10% serum, suggesting that the fluorescent probe resided exclusively in the outer leaflet of the plasma membrane at 2°C.
After insertion into the plasma membrane, internalization of (palmitoyl, Ce-NBD)-PC occurred when the cells were warmed to 37°C. This resulted in four different labeling patterns: (1) little or no internalization of (palmitoyl, C6-NBD)-PC into punctate vesicles was observed in Vero cells. (2) Transport of (palmitoyl, C6-NBD)-PC to the region of the Golgi apparatus and to a small number of intracellular vesicles was observed in both V79 and CHO-K1 cell lines. (3) A large number of fluorescently labeled intracellular vesicles with little or no labeling in the region of the Golgi apparatus appeared after the internalization of (palmitoyl, C6-NBD)-PC in BHK, CHO-15B, MDCK and WI-38 cell lines. (4) Accumulation of (palmitoyl, C6-NBD)-PC in small vesicles, mitochondria and the nuclear envelope was observed in VA-2 cells. In addition, cells having a defect in glycoprotein processing and those transformed with simian virus 40 (SV40) internalized the fluorescent lipid probe differently compared with parental lines. Neither differences in rates of endocytosis nor rates of (palmitoyl, C6-NBD)-PC degradation between cell types appears to cause the observed dissimilarities in intracellular lipid transport. We suggest that these different cell types may have dissimilar pathways of intracellular lipid trafficking or differential regulation of a common transport pathway, and that the predominant pathway of lipid translocation can be altered in cells by changing the composition of their glycoproteins or by viral transformation.
Changes in nuclear distribution of large tumor antigen correlate with the proliferative activity of simian virus 40-transformed cells
Authors: Ordenes Gamaliel E. , Beck Ingrid , Carmona Maria T. , Santos Myriam 
Published: April 2021

ABSTRACT
A correlation between intranuclear distribution of large tumor antigen (T-ag) and proliferative activity in Simian virus 40 (SV40)-transformed cells has been established. Nuclear T-ag was cytochemically detected by the immunoperoxidase reaction. Cell populations with a high growth rate displayed a heterogeneous pattern with patches of positive reaction surrounded by large and irregular negative areas. By contrast, cell populations with a low growth rate presented a rather homogeneous distribution of nuclear T-ag with a finely granular positive reaction all over the nucleus, excluding nucleolus-like areas. The latter pattern was also observed in cells that had been inhibited from traversing S phase. In this case, the heterogeneous distribution of T-ag within the nucleus was recovered once the cells were allowed to proceed again through S phase, or through G2 phase. Highly condensed chromatin in mitotic chromosomes appeared to exclude T-ag fully as early as in prophase. Then, nuclear distribution of T-ag changes throughout the cell cycle, thus suggesting it might be a cell cycle-dependent event. Interestingly, thymidine-labeled cells from cultures with a high growth rate presented a heterogeneous distribution of autoradiographic grains. Regardless of their mechanism(s) of origin, T-ag-enriched areas might correspond to nuclear sites where a specific function, such as DNA synthesis, is being carried out.
5-Azacytidine-induced alterations in the GH12C1 cells: effects on cellular morphology, chromosome structure, DNA and protein synthesis
Authors: Parrow Vendela C. , Aleström Peter , Gautvik Kaare M. 
Published: April 2021

ABSTRACT
Growth hormone-producing rat pituitary tumor cells (GH12C1) were cultivated in the presence of 5-azacytidine (5-AC). After cessation of treatment the cells were allowed to recover in normal cell culture medium prior to subcultivation. Within hours after the subcultivation the cells underwent several morphological alterations, later followed by changes in growth pattern: (1) cell shape was irreversibly changed from round or spindle shape to different clones of fibroblast-like cells; (2) some of these clones later formed foci; and (3) an extensive generation of multinucleated cells was seen. The demethylation was maximal approximately one week after the first subcultivation in the absence of 5-AC.
Severe alterations of the chromosome structure were seen after the first subcultivation. During the following weeks the 5-AC-treated cells showed impaired chromosome condensation, and homogeneous staining with quinacrine mustard. During this period the DNA synthesis is doubled when compared to control cell DNA synthesis. The core histone synthesis increases in parallel with the DNA synthesis, but the majority of nuclear proteins, including the linker histones, remain at the control level. This results in a ratio of linker histone to core histone synthesis at approximately half the control cell level. The altered ratio of synthesis slowly decreased to control levels during a period of five weeks of continuous cultivation in the absence of the drug, and the under-condensed chromosomes could no longer be seen. The induced novel phenotypes with their pleiomorphic appearances were conserved.
The growth hormone synthesis remained constant during all phases of the experiment and prolactin synthesis was not induced.
Inositol tris- and polyphosphate formation during chemotaxis of Dictyostelium
Authors: Europe-Finner G. N. , Gammon B. , Wood C. A. , Newell P. C. 
Published: April 2021

ABSTRACTUsing the technique of HPLC with Partisil SAX columns, we have found that stimulation of amoebae of Dictyostelium discoideum with the chemoattractant cyclic AMP induces the rapid accumulation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), with a peak at 5s. A smaller HPLC peak (designated P3) that elutes just after the Ins(1,4,5)P3 peak accumulates more slowly to a maximum at 20 s. In control studies, the changes in Ins(1,4,5)P3 were shown not to be due to varying recovery from the cell extracts and a comparison of reverse-phase and Partisil SAX HPLC columns showed similar values for determinations by either method.The involvement of a G-protein in this chemotactic system was confirmed by the finding that accumulation of Ins(1,4,5)P3 was elicited by the addition of GTP γS (5′-[gamma-thio]triphosphate) to saponin-permeabilized amoebae.A study of the changes in the lipid-soluble phosphatidyl inositol phosphates demonstrated that cyclic AMP also stimulated a rapid loss of radioactivity from 32P-labelled phosphatidylinositol 4,5-bisphos-phate (PtdIns(4,5)P2), which corresponded in its timing to the rise in Ins(1,4,5)P3, indicating that a phosphoinositidase C (phospholipase C) is present that can be stimulated by occupation of the cell surface cyclic AMP receptors.
Studies on the mechanism of embryonic frog nerve orientation in a small applied electric field
Authors: Mccaig Colin D. 
Published: April 2021

ABSTRACT
The mechanism of nerve orientation in an applied electric field has been investigated using a number of pharmacological agents. Galvanotropism may depend on redistribution within the plasma membrane of integral membrane proteins (IMP); blocking this with concanavalin A inhibited orientation. Orientation may depend also on an influx of Ca2+; Co2+ and La3+ blockade of calcium channels inhibited turning in an electric field. Organic blockers of calcium channels did not influence orientation, suggesting that L-type Ca2+ channels may not be present at the growth cone. Procedures that may induce asymmetric entry of Ca2+ on the anodal side of cells caused a reversal of normal galvanotropism, with growth directed towards the anode. This may implicate local levels of cytoplas mic Ca2+ within the growth cone in controlling turning behaviour. An asymmetric distribution of filopodia precedes and may predict the direction of nerve growth in an electric field. Various pharmacological agents perturbed the distribution of filopodia in such a way that this did not reflect subsequent orientation. It is suggested that, normally, local Ca2+ increases and an asymmetry of filopodia operate together in determining orientation, but that filopodial activity is subordinate to and can be overriden by local Ca2+ levels in the growth cone. In addition, two of the drug treatments markedly increased rates of nerve growth, which may be of importance in nerve regeneration.
Anaphase onset and dephosphorylation of mitotic phosphoproteins occur concomitantly
Authors: Vandré Dale D. , Borisy Gary G. 
Published: April 2021

ABSTRACT
The cyclical phosphorylation and dephosphorylation of the centrosome during mitosis was analyzed by immunofluorescence methods using the MPM-2 antibody, which reacts with a subset of mitotic phosphoproteins. Quantification of MPM-reactivity indicated that centrosomal phosphorylation attained a maximal level just prior to ana-phase onset. This level was maintained in meta-phase cells blocked from further mitotic progression with the microtubule depolymerizing agent nocodazole. However, when nocodazole was added to cells that had just initiated anaphase, the level of centrosomal phosphorylation decreased rapidly as in untreated anaphase cells. We conclude that the onset of dephosphorylation of the centrosome coincided with the onset of anaphase and continued in the absence of chromosome movement. Dephosphorylation of MPM-2 reactive phos-phoproteins may be taken as a biochemical indicator of anaphase onset.
Circadian changes in cyclic amp levels in synchronously dividing and stationary-phase cultures of the achlorophyllous ZC mutant of Euglena gracilis
Authors: Carré Isabelle A. , Laval-martin Danielle l. , Edmunds Leland n. 
Published: April 2021

ABSTRACT
Oscillations in adenosine 3′,5′-cyclic monophos-phate (cyclic AMP) level have been proposed to be part of the biochemical feed-back 100p(s), or *clock(s)’, believed to underlie circadian rhythmicity. This possibility has been examined for a cellular circadian oscillator in synchronously dividing (or nondividing) cultures of the photosynthesis-deficient ZC mutant of the alga Euglena gracilis Klebs (Z). We have demonstrated a bimo-dal, autonomously oscillating, circadian variation of cyclic AMP content in this unicell. Rhythmic changes of the cyclic AMP level, which may reflect the transition of the cell population through the different phases of the cell division cycle (CDC) in division-phased cultures, also persisted after the culture medium had become limiting and the cells had stopped dividing. We have also shown that the free-running, circadian oscillation of cyclic AMP content displayed by nondividing cells in continuous darkness could be phase-shifted by a light signal (a property inherent to most circadian systems), in a manner that could be predicted from the phase-response curve previously obtained for the cell division rhythm in the ZC mutant. These results suggest a possible role for cyclic AMP, either as an element of the coupling pathway for the control of the CDC by the circadian oscillator, or as a ‘gear’ of the clock itself.
Characterization of mitotic motors by their relative sensitivity to amp-pnp
Authors: Lee Greta Marlene 
Published: April 2021

ABSTRACT
The relative sensitivities of the motors for mitotic chromosome movements and saltatory motion were compared using a nonhydrolyzable analog of ATP, AMP-PNP. K+AMP-PNP was microinjected into PtKl cells at the time of nuclear envelope disassembly or at anaphase onset. To produce a dose-response curve for the effect of AMP-PNP on the rate of movement, the intracellular concentration of AMP-PNP in individual cells was measured. The volume injected into each cell was determined by adding dextrans labeled with Lucifer Yellow to the injection buffer, measuring the injected cell’s fluor-escence intensity, and then comparing the value with the fluorescence intensity of known volumes of Lucifer Yellow dextran solution. AMP-PNP produced a 50% inhibition of spindle elongation at 0.2 mM, of saltatory motion at 0.8 mM, and of chromosome movement at 8.6 mM. Prometaphase chromosome movement and anaphase chromosome-to-pole movement were similarly inhibited by AMP-PNP. Equivalent volumes of injection buffer containing 1% Lucifer Yellow dextran had no effect on chromosome movement, spindle elongation or saltatory motion. Although AMP-PNP occasionally produced shorter anaphase spindles, tubulin immunofluorescence revealed the presence of abundant spindle microtubules. Metaphase cells treated with very high cell concentrations of AMP-PNP had spindles with unusually long astral microtubules; thus microtubules are stabilized rather than broken down by AMP-PNP. In conclusion, spindle elongation is four times more sensitive than saltatory motion to AMP-PNP and 40 times more sensitive than chromosome movement. When these sensitivities to AMP-PNP are considered with the results from other studies, it can be concluded that the molecular motors for spindle elongation, chromosome movement and saltatory motion are different.
Measurement of volume injected into individual cells by quantitative fluorescence microscopy
Authors: Lee Greta Marlene 
Published: April 2021

ABSTRACT
Pressure microinjection is frequently used to introduce substances into mammalian cells, but precise quantitation of the volume injected into individual cells has been difficult. A simple and reliable procedure for determining the volume injected was developed in order to determine what intracellular concentration of AMP-PNP was necessary to inhibit specific cellular processes. The technique uses fluorescent Lucifer Yellow-labeled dextrans in the microinjection buffer and quantitative fluorescence microscopy to measure the fluorescence intensity of the injected cell. The volume injected is computed from a standard curve derived from the volume and fluorescence of spherical, microscopic droplets of Lucifer Yellow dextran solution. The droplets are ejected from a micropipet into immersion oil where they sink to rest on a siliconized coverslip. For the measurement of fluorescence, an inexpensive photomultiplier system that is attached to a fluorescence microscope is described. The potential uses of this method for other microassays are discussed.
Polymorphism of tubulin assembly in vitro
Authors: Karecla Paula , Hirst Elizabeth , Bayley Peter 
Published: April 2021

ABSTRACT
Polymorphism in the self-assembly of tubulin dimer and microtubule protein (tubulin plus the microtubule-associated proteins) has been investigated as a function of systematic variation of solution composition (i.e. buffer ion, [glycerol] and [Mg2+]). The nature of the assembly product was examined using negative staining and thin sectioning electron microscopy.
The morphology of the product of assembly of tubulin dimer was found to be strongly influenced by the concentration of glycerol and Mg2+ in Pipes and Mes buffers; the effects are less marked in phosphate buffer. Formation of bona fide micro-tubules in 0.1 M-Pipes occurs for a limited range of solution conditions (e.g. with [glycerol] &lt;2 M and [Mg2+]&lt;l mM). Conditions of elevated [glycerol] and [Mg2+], which enhance the rate and extent of assembly, have the adverse effect of strongly promoting the formation of polymorphic forms in addition to, and in place of, the normal microtubule morphology.
In both Pipes and Mes buffers, increasing [glycerol] from 1 to 3 M favours the formation of extended multiply curved sheets, apparently made up from a basic structure with an S-like cross-section. By contrast, increasing [Mg2+] promotes the formation of junctions between microtubule walls, giving products whose cross-section shows multiple hook-like appendages, attached to closed microtubules. The assembly of tubulin dimer in a typical ‘dimer assembly buffer’ (e.g. 0.05-0.1 M-Mes, with 1-3.4M-glycerol and 2–7mM-Mg2+), invariably produces substantial proportions of non-microtubule structures such as open sheets, ribbons, and hooked structures.
We conclude that the self-assembly of tubulin dimer exclusively into bona fide microtubules occurs over a very restricted range of solution conditions in the normally used Pipes- and Mes-based buffers. Deviation from these conditions readily promotes the formation of mixtures of polymorphic forms. Many buffer systems used for the assembly and disassembly of microtubules composed of tubulin dimer appear likely to promote the formation of structures related to, but significantly different from, normal microtubules. This represents a cautionary factor in the interpretation of in vitro assembly and disassembly properties of microtubules.
A novel component of the axonal cortical cytoskeleton, A60, defined by a monoclonal antibody
Authors: Rayner Denise A. , Baines Anthony J. 
Published: April 2021

ABSTRACT
A Mr 60 000 protein of the axonal cortical cytoplasm, which is recognized by a novel monoclonal antibody, is described. The antibody, DR1, was produced by immunizing mice with a soluble extract of bovine brain membranes that is enriched in known membrane cytoskeletal proteins. DR1 recognizes a Mr60000 protein in this extract. Immunofluorescence and subcellular fractionation reveal that the protein is primarily located in axons, where it appears to form a thick lining to the axolemma. Operationally, this Mr 60000 protein is defined as a cytoskeleton-associated peripheral membrane protein. It is solubilized from brain membranes only under harsh conditions (0.1M-NaOH), but not with KI (0.8M) or Triton X-100 (1%). It is present at higher levels in the central nervous system than in peripheral nerves that have been examined. The Mr 60 000 protein copurifies with neurofilaments through cycles of assembly and disassembly. It does not appear to react with the anti-IFA antibody, suggesting that it is not a member of the intermediate filament class of proteins. This Mr60000 protein, which we designate A60, is distinct from other known neurofilament-associated proteins, including the Mr 60 000 protein α-internexin and the Mr 58000 intermediate filament protein peripherin. A60 is suggested as being a previously unrecognized component of the axonal cortical cytoskeleton.
Regulatory signals affecting a selective loss of mRNA in Dictyostelium discoideum
Authors: Hassanain Hamdy H. , Kopachik Will 
Published: April 2021

ABSTRACT
We identified signals that affect mRNA levels complementary to a gene that is highly expressed in vegetative Dictyostelium discoideum cells. This gene has been cloned as cDNA in the plasmid pcD-D2. The level of transcripts homologous to pcD-D2 fell dramatically in strain XP55 during the aggregation stage of development when cells differentiate on agar. The level, however, did not fall simply as a result of starvation or aggregationspecific cell contact. Rather, before the level is reduced cells must be deprived of amino acids and cyclic AMP administered in amounts and at intervals in pulses to mimic cyclic AMP signal-relay in aggregation. This effect can be blocked either with cyclic AMP-S (a non-hydrolysable cyclic AMP analogue) or adenosine, both of which prevent cyclic AMP binding to the cyclic AMP cell surface receptor. It is also blocked in ‘frigid’ aggregationdeficient mutants HC85 and HC112 known to be defective in a Gα protein. We conclude that the transcript level is balanced by positive nutritional signals acting against negative signals transduced in part through a cell surface cyclic AMP receptor.
Differences between adult and foetal fibroblasts in the regulation of hyaluronate synthesis: correlation with migratory activity
Authors: John Chen W. Y. , Grant Michael E. , Schor Ana M. , Schor Seth L. 
Published: April 2021

ABSTRACT
We have previously reported that confluent foetal fibroblasts migrate into three-dimensional collagen gel matrices to a significantly greater extent than do adult cells. Hyaluronic acid (HA) is a major constituent of the extracellular matrix deposited by fibroblasts and has been demonstrated to stimulate the migration of a number of different cell types. Previous studies have indicated that the synthesis of HA by normal adult skin fibroblasts declines significantly when the cells achieve confluence. Data presented in this paper indicate that foetal fibroblasts differ from adult cells in this respect, in that they do not show an inverse relationship between cell density and HA synthesis, i.e. confluent foetal fibroblasts continue to produce approximately the same amount of HA as do subconfluent cells. These data suggest that the synthesis of relatively high levels of HA by foetal fibroblasts at confluence may be causally related to the elevated migration displayed by these cells. In this context, a close correlation was observed between the level of HA synthesized by confluent foetal and adult fibroblasts and the differential migratory activity displayed by these cells. Such differences in HA synthesis and migratory behaviour were only apparent at cell confluence, with subconfluent foetal and adult fi-broblasts being indistinguishable in terms of these two criteria. Our data further reveal that: (1) cell density affects the size class of HA synthesized by both foetal and adult cells; and that (2) there is a considerable degree of heterogeneity amongst the nine different fibroblast lines examined in this study in terms of the size class of HA that they produce.