bluuebunny/crossref_metadata_2025_split huggingface dataset:
The pericentriolar lattice of PtK2 cells exhibits temperature and calcium modulated behavior
Authors: Baron Andre T. , Suman Vera J. , Nemeth Edward , Salisbury Jeffrey L. 
Published: April 2021

ABSTRACT
In this study, we demonstrate that manipulations of temperature and free calcium alter the morphology of the centrin-containing pericentriolar lattice of PtK2 cells. Immunofluorescence microscopy reveals that low-temperature incubation (4°C) causes anti-centrin-labeled pericentrosomal spots to coalesce in the peripheral cytoplasm, and fuses small spots into larger spots near the cell center. At electron microscopic resolution, well-formed pericentriolar satellites appear around the centrioles in response to incubation at 4°C. Elevated free calcium enhances these low-temperature-dependent effects. The data suggest that peri-centrosomal spots correspond to one or more pericentriolar satellites, and that pericentriolar satellites and centrosomal matrix are interconvertable forms of the same material. Transient elevation of intracellular free calcium at 37°C from a basal level of 3.7×10−8 M to a peak level of 2.0×10−7 M within 30 seconds with ionomycin results in a 35% increase in pericentrosomal spot number throughout the cytoplasm. The number of pericentrosomal spots is 50% larger 2 minutes after ionomycin addition; these spots are also nearer to the cell center as compared to 30 seconds after ionomycin addition. As intracellular free calcium returns to a basal level over 5 minutes, the number of spots and their cellular distribution resume a pretreatment value and pattern. We interpret these observations to indicate movement of pericentrosomal spots toward the cell center in response to the flux in intracellular free calcium. Alternatively, it is possible that no movement has occurred, but that the rise in free calcium has unmasked an epitope responsive to our anti-centrin antiserum. Regardless of the interpretation, we conclude that the pericentriolar lattice exhibits calcium-modulated behavior.
Evidence that kinetochore microtubules in crane-fly spermatocytes disassemble during anaphase primarily at the poleward end
Authors: Wilson Paula J. , Forer Arthur , Leggiadro Cindy 
Published: April 2021

ABSTRACT
Anaphase chromosome motion involves the disassembly of kinetochore microtubules. We wished to determine the site of kinetochore microtubule disassembly during anaphase in crane-fly spermatocytes.
In crane-fly spermatocyte spindles, monoclonal antibody 6-11B-1 to acetylated α-tubulin labels kinetochore microtubules almost exclusively, with an area immediately adjacent to the kinetochore being weakly or not labelled. This ‘gap’ in acetylation at the kinetochore serves as a natural marker of kinetochore microtubules in the kinetochore fibre. We measured the length of the gap on kinetochore fibres in metaphase and anaphase in order to deduce the fate of the gap during anaphase; we used this information to determine where kinetochore microtubules disassemble in anaphase. Gap lengths were measured from confocal microscope images of fixed spermatocytes dual labelled with 6-11B-1 to acetylated α-tubulin and YL1/2 to tyrosinated α-tubulin, the latter being used to determine the positions of kinetochores.
In metaphase the average gap length was 1.7 µm. In anaphase, the gap appeared to decrease in length abruptly by about 0.4 µm, after which it decreased in length by about 0.2 µm for every 1 µm that the chromosome moved poleward. PacMan models of chromosome movement predict that this ‘gap’ in staining should disappear in anaphase at a rate equal to that of chromosome movement. Thus, our results do not support theories of chromosome motion that require disassembly solely at the kinetochore; rather, in crane-fly spermatocytes kinetochore microtubule disassembly in anaphase seems to take place primarily at the poles.
The effects of selective inhibitors of matrix metalloproteinases (MMPs) on bone resorption and the identification of MMPs and TIMP-1 in isolated osteoclasts
Authors: Hill Peter A. , Murphy Gillian , Docherty Andrew J. P. , Hembry Rosalind M. , Millican T. Andrew , Reynolds John J. , Meikle Murray C. 
Published: April 2021

ABSTRACT
We have compared the effects of a general matrix metalloproteinase (MMP) inhibitor (CT435) with those of a concentration-dependent specific gelatinase inhibitor (CT543; Ki&lt;20 nM) on bone resorption in vitro. The test systems consisted of measuring: (i) the release of 45Ca2+ from prelabelled mouse calvarial explants; (ii) the release of 45Ca2+ from prelabelled osteoid-free calvarial explants cocultured with purified chicken osteoclasts; and (iii) lacunar resorption by isolated rat osteoclasts cultured on ivory slices.
Both CT435 and CT543 dose-dependently inhibited the release of 45Ca2+ from neonatal calvarial bones stimulated by either parathyroid hormone or 1,25-dihydroxyvitamin D3. Moreover, CT543 produced a 40% inhibition at a concentration (10—8 M) selective for the inhibition of human gelatinases A and B. CT435 (10−5 M) and CT543 (10−5 M) partially inhibited the release of 45Ca2+ from osteoid-free calvarial explants by chicken osteoclasts with a maximum of approximately 25% for unstimulated cultures, and approximately 36% for cultures stimulated by interleukin-1α (IL-1α; 10−10 M). Neither inhibitor prevented lacunar resorption on ivory by unstimulated rat osteoclasts, but the compounds produced a partial reduction in both the number and total surface area of lacunae in IL-1α-stimulated cultures, with maximal action at 10−5 M. Neither of the inhibitors affected protein or DNA synthesis, nor the IL-1α-stimulated secretion of the lysosomal enzyme β-glu-curonidase.
Immunocytochemistry demonstrated that isolated rabbit osteoclasts constitutively expressed gelatinase A and synthesized gelatinase B, collagenase and stromelysin, as well as the tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) following IL-1α stimulation.
These experiments have shown that in addition to collagenase, gelatinases A and B are likely to play a significant role in bone resorption. They further suggest that MMPs produced by osteoclasts are released into the sub-osteoclastic resorption zone where they participate in bone collagen degradation.
Replicon clusters may form structurally stable complexes of chromatin and chromosomes
Authors: Sparvoli Elio , Levi Marisa , Rossi Emanuele 
Published: April 2021

ABSTRACT
Nuclear DNA replication was monitored ‘in situ’ in pea nuclei with the bromodeoxyuridine antibody technique. The labelling appeared to be restricted to a number of finely distinct spots. The labelling was followed through three subsequent cell cycles in meristematic and differentiating pea root cells. The results show that the spots as seen just after the labelling persist distinctly over the mitotic chromosomes as well as in the nuclei of the following cell cycles up to 44 hours after the pulse. Moreover, they are also present in the nuclei of differentiating cells. The spots over the mitotic chromosomes in specific cases give rise to a dynamic banding. Nuclei of the second and third cycle show absence of labelling in specific zones, owing to the segregation of the labelled strands of chromosomal DNA. The maintenance of the spotted appearance of the replication clusters through all stages of the three subsequent cell cycles may be an indication in favour of the hypothesis that such clusters represent structurally stable replicon complexes held together by the nuclear matrix and the chromosome scaffold.
Sequence analysis of MAP2 function in living cells
Authors: Ferralli Jacqueline , Doll Thierry , Matus Andrew 
Published: April 2021

ABSTRACT
Microtubule-associated protein 2 (MAP2) is an abundant neuron-specific protein that binds to microtubules through a domain near its carboxyl terminus that contains either three or four similar repeats of a 31 amino acid motif. When expressed in non-neuronal cells by transfection MAP2 stabilises microtubules and induces their rearrangement into long bundles that are capable of supporting process outgrowth. To investigate which elements in the MAP2 sequence are involved in these functions we have constructed a series of deletion mutants of the short embryonic form of MAP2, MAP2c, and transfected them into non-neuronal cells. This showed that the strength of binding to microtubules increased with the number of repeats present in the construct. However, the repeat domain itself was insufficient for microtubule binding, which required in addition contiguous sequences either amino-terminal or carboxyl-terminal to the repeats themselves. Particularly on the amino-terminal side of the repeats, where there is a proline-rich domain, step-wise increases in the length of neighbouring sequence produced a gradual increase in microtubule binding. The apparent strength of binding to microtubules produced by mutant MAP2 forms was further correlated with the degree of bundling they induced as well as with the ability of the resulting microtubules to support process outgrowth. These results indicate that the interaction of MAP2 with microtubules is mediated by the combined action of several weak binding sites, including each of the repeat motifs and elements in the sequences on either side of them, whose additive effect produces the strong binding of the native MAP2 molecule. The results further indicate that both the bundling and stiffening of microtubules by MAP2 are correlated with the strength of its binding to them and suggest that these properties are a direct result of microtubule stabilisation.
Dispersive locomotion of human neutrophils in response to a steroid-induced factor from monocytes
Authors: Chettibi S. , Lawrence A. J. , Young J. D. , Lawrence P. D. , Stevenson R. D. 
Published: April 2021

ABSTRACT
A monocyte-derived factor that stimulates the locomotion of human neutrophils on an albumin-coated glass surface has been prepared from the culture supernatant of dexamethasone-treated human monocytes and called STMS (steroid-treated monocyte supernatant). A modified cell tracking program has been developed and the parameters of locomotion determined by the analysis of Gail and Boone for cells moving in a persistent random walk. Cells moving in uniform concentrations of STMS, interleukin- 8 (IL-8) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) chosen to give a sub-maximal speed of locomotion show persistent, random and constrained random diffusion, respectively, with augmented diffusion coefficients of 0.8±0.1, 0.14±0.02 and 0.12±0.03 µm2 per second for STMS, IL-8 and fMLP, respectively. The augmented diffusion coefficient and the underlying persistence are therefore sensitive quantitative assay parameters for STMS activity and the qualitative characteristics of locomotion allow STMS activity to be distinguished from that of all other factors tested. The contribution of lowered adhesion to locomotion was examined in a novel tilt-assay, which demonstrated that cells in the presence of STMS, but not other factors, moved down slope with significantly increased speed while maintaining contact with the substratum. The results were interpreted in terms of the bipolar form of STMS-treated cells, contrasting with multipolar forms in response to other agents. This together with low adhesiveness plus an inherent tendency of a single locomotor focus to continue motion in its original direction has been used to explain the difference between response to STMS and other factors. STMS has been proposed either to prevent directed locomotion of neutrophils to an inflammatory site or to promote dispersive locomotion away from such a site and perhaps to inhibit neutrophil transmigration between endothelial cells.
Transport of a lysosomal membrane glycoprotein from the Golgi to endosomes and lysosomes via the cell surface in African trypanosomes
Authors: Brickman Marla Jo , Balber Andrew E. 
Published: April 2021

ABSTRACT
gp57/42 is a membrane glycoprotein localized in the trans - Golgi, flagellar pocket region of the cell surface, endosomes and lysosomes of bloodstream forms of Trypanosoma brucei rhodesiense. Pulse-chase immunoprecipitation experiments revealed that gp57/42 acquires a unique N-linked oligosaccharide recognized by the CB1 monoclonal antibody 20-30 minutes after protein synthesis, probably in the trans - Golgi. We refer to gp57/42 molecules that carry the CB1 eptiope as CB1-gp. Pulse labeled CB1-gp contained only one core protein, p57, when chase times were 30 minutes or less. As time of chase increased from 30 to 60 minutes, a new polypeptide, p42, appeared in N-glycanase-treated CB1 immunoprecipitates. Since p57 and p42 share 10 of 13 methionyl peptides, we conclude that p42 is a fragment of p57. Cleavage of p57 to p42 was not inhibited when cells were chased in two thiol protease inhibitors or in 3,4-diiso-coumarin, but was inhibited by leupeptin. Cell surface biotinylation was used to determine if newly synthesized CB1-gp was transported from the Golgi to the surface. When cells were pulse labeled and chased for 30 minutes, as much as 40% of the radiolabeled CB1-gp could be biotinylated on the cell surface. The amount of CB1-gp that could be biotinylated decreased when chases were extended from 30 to 60 minutes, suggesting that pulse labeled CB1-gp left the surface. In contrast, pulse labeled variant surface glycoprotein molecules continued to accumulate on the surface where they could be biotinylated between 30 and 60 minutes of chase. Biotinylated CB1-gp derived from cells chased for 30 minutes contained p57 but no p42. However, when labeled cells were biotinylated after a 30 minute chase and then incubated another 30 minutes at 37°C, the biotinylated CB1-gp contained both p57 and p42. The p57 in biotinylated CB1-gp was not cleaved to p42 if the additional incubation was done at 4 or 12°C. This suggests that transport to a compartment where processing occurs and/or the processing enzymes are inhibited by low temperature. When surface biotinylation was done after a 60 minute chase, p42 was detected in biotinylated CB1-gp, suggesting that CB1-gp molecules had passed through the processing compartment and then appeared on the cell surface. Thus, a major portion of the newly synthesized CB1-gp is routed from the Golgi to endocytic compartments via the cell surface. In trypanosomes this process involves a unique surface domain, the flagellar pocket. Otherwise, this pathway of lysosomal membrane glycoprotein transport has been conserved in evolution from the trypanosomes to the vertebrates.
Functional consequences in yeast of single-residue alterations in a consensus calmodulin
Authors: Harris Eva , Watterson D. Martin , Thorner Jeremy 
Published: April 2021

ABSTRACT
A synthetic gene encoding a ‘consensus’ calmodulin (synCaM) was able to substitute for the Saccharomyces cerevisiae calmodulin gene (CMD1), even though synCaM is only 60% identical in primary amino acid sequence to yeast Cmd1. Twelve different synCaM mutants were also expressed in yeast. Seven of the 12 mutant synCaMs supported germination and growth of Cmd1-deficient spores. Five of the 12 mutant synCaMs were incapable of supporting germination of Cmd1-deficient spores and, of these, four were also incapable of supporting vegetative growth of Cmd1-deficient haploid cells. The five nonfunctional synCaM mutants were expressed at levels equivalent to, or higher than, the seven synCaM mutants that were able to substitute for Cmd1; thus, the inability to function was not simply due to inadequate expression or rapid degradation. All nonfunctional synCaM mutants shared a single charge reversal mutation in the central helix (E84K), which was found to be sufficient to confer the lethal phenotype. The ability of another mutant synCaM (S101F) to support growth of Cmd1-deficient cells was dependent on cell ploidy. Another mutant (K115Y) supported spore germination and vegetative growth, but not meiosis and sporulation. The terminal phenotype of cells lacking a functional calmodulin included a dramatic accumulation of polymerized microtubules.
Inductive effect of copper deficiency on the reversion of dedifferentiated rat hepatoma cells and on gene amplification
Authors: Renault Eric , Deschatrette Jean 
Published: April 2021

ABSTRACT
Cells of a dedifferentiated rat hepatoma clone were submitted in vitro to copper deficiency. This treatment caused inhibition of cell growth. In addition, in treated cultures, the frequency of differentiated revertants selected in glucose-free medium was drastically increased when compared with the spontaneous frequency. The maximum effect was observed when cell proliferation spontaneously resumed after 20 days of copper deficiency. Furthermore, a copper depletion/replenishment protocol applied before the selection of revertants reduced the period of time of copper deficiency that was necessary to provoke the reversion process.
It has been previously demonstrated that cell growth arrest and reinitiation may induce gene amplification events. Amplification of the dihydrofolate reductase gene as an indicator of such events was tested during the copper deficiency treatment. The frequency of cells resistant to increasing methotrexate concentrations due to gene amplification was enhanced by the treatment, just as was the frequency of differentiated revertants. These results suggest that in rat hepatoma cells the phenotypic transition to the stable differentiated state involves gene amplification and/or genome rearrangement.
ATP depletion: a novel method to study junctional properties in epithelial tissues: cytoskeleton: I. Rearrangement of the actin
Authors: Bacallao Robert , Garfinkel Alan , Monke Steven , Zampighi Guido , Mandel Lazaro J. 
Published: April 2021

ABSTRACT
The effect of cellular injury caused by depletion of intracellular ATP stores was studied in the Madin-Darby canine kidney (MDCK) and JTC cell lines. In prior studies, it was shown that ATP depletion uncouples the gate and fence functions of the tight junction. This paper extends these observations by studying the changes in the actin cytoskeleton and tight junction using electron microscopy and confocal fluorescence microscopy in combination with computer-aided three-dimensional reconstruction. Marked regional differences in the sensitivity to the effects of ATP depletion were observed in the actin cytoskeleton. Actin depolymerization appears to first affect the cortical actin network running along the apical basal axis of the cell. The next actin network that is disrupted is the stress fibers found at the basal surface of the cell. Finally, the actin ring at the level of the zonulae occludens and adherens is compromised. The breakup of the actin ring correlates with ultrastructural changes in tight junction strands and the loss of the tight junction’s role as a molecular fence. During the process of actin network dissolution, polymerized actin aggregates form in the cytoplasm. The changes in the junctional complexes and the potential to reverse the ATP depletion suggest that this may be a useful method to study junctional complex formation and its relationship to the actin cytoskeletal network.
ATP depletion: a novel method to study junctional properties in epithelial tissues: II. Internalization of Na+, K+-ATPase E-cadherin
Authors: Mandel Lazaro J. , Doctor R. Brian , Bacallao Robert 
Published: April 2021

ABSTRACT
MDCK and JTC cells were subjected to ATP depletion by treating the cells with 10 μM antimycin A and 10 mM 2-deoxyglucose. As visualized by confocal fluorescence microscopy, E-cadherin and Na+,K+-ATPase were rapidly internalized following depletion of the intracellular ATP stores. The time course of internalization was similar to the depolymerization of the cortical actin network and dissolution of the actin ring (see companion paper, this volume, pp. 3301-3313). Cell surface biotinylation was used to assay the amount of surface-accessible E-cadherin and Na+,K+-ATPase during ATP depletion. At 30 minutes of ATP depletion, 74% and 69% of E-cadherin and Na+,K+-ATPase were internalized, respectively, in MDCK cells. By 60 minutes of ATP depletion, internalization increased to 95% and 89%, respectively. The redistribution of both plasma membrane proteins was not microtubule dependent. Similar results were observed in JTC cells. Total biotinylated protein decreased by 67% and 82%, after 30 minutes and 60 minutes of ATP depletion, respectively. The E-cadherin internalization strongly suggests that disruption of adherens junctions occurred following ATP depletion. These results, along with the previously described loss of tight junction integrity, suggest that ATP depletion may be a useful method to study the assembly and disassembly of junctional complexes in epithelial cells.
A group of integral membrane proteins of the rat liver Golgi contains a conserved protein of 100 kDa
Authors: Pryde James G. 
Published: April 2021

ABSTRACT
Rat liver Golgi membranes were washed with KCl and urea, and a polyclonal antiserum that stained the Golgi complex by immunofluorescence microscopy was raised. A group of proteins of apparent molecular mass 500 kDa, 200 kDa and 100 kDa were identified by immunoblotting with the antiserum, and were enriched in the Golgi membrane fraction. These proteins were also localised to the Golgi by immunofluorescence microscopy with affinity-purified antibodies. They are integral membrane proteins, and protease digestion experiments suggested that they are not exposed on the cytoplasmic face of the Golgi membrane.
Immunofluorescence microscopy showed that staining of the Golgi complex by antibodies to the 100 kDa Golgi protein can be demonstrated among a wide range of mammalian species. This conservation may point to an important structural or functional role for the molecule. When the 100 kDa protein was reduced with dithiothreitol it was no longer recognised by the anti-Golgi antiserum. During phase separation in Triton X-114 the 100 kDa protein partitioned into the aqueous phase, rather than into the detergent phase, suggesting that it has a large luminal domain of hydrophilic amino acids.
The protein phosphatase inhibitor okadaic acid induces defects in cytokinesis and organellar genome segregation in Trypanosoma brucei
Authors: Das Arpita , Gale Michael , Carter Victoria , Parsons Marilyn 
Published: April 2021

ABSTRACT
Mitosis and cytokinesis are events that are highly coordinated in most eukaryotic cell cycles. African trypanosomes possess a single mitochondrion and must additionally coordinate the organellar division cycle. Here we report that okadaic acid, a potent and specific inhibitor of protein phosphatases PP1and PP2A, uncouples these cycles in living trypanosomes. Cell cycle analysis of treated cells revealed elevated DNA content. Microscopic examination indicated that okadaic acid treatment yielded multinucleate cells with a single mitochondrial network indicating these cells have undergone mitosis but failed to complete cytokinesis. Immunofluorescence analysis of 5-bromo-2deoxyuridine incorporation demonstrated that the mitochondrial DNA was replicated but did not segregate. The dose response curve for inhibition of the normal cell cycle paralleled that for the in vitro inhibition of protein phosphatase activities with IC50s of approximately 20 nM okadaic acid. These results suggest the involvement of a PP1/PP2A-like activity in coordinating mitosis, mitochondrial DNA division and cytokinesis in trypanosomes.