bluuebunny/crossref_metadata_2025_split huggingface dataset:
Protein myristoylation in human mononuclear phagocytes: modulation by interferon-y and tumor necrosis factor
Authors: Poli Giovanni , Sorio Claudio , Berton Giorgio 
Published: April 2021

ABSTRACT
Labelling of cells with [3H]myristic acid and analysis of labelled proteins by SDS-PAGE and fluorography, enabled the identification of a limited number of myristoylated proteins in human monocytes and monocyte-derived macrophages. In human monocytes, cultivated for one to three days, major myristoylated proteins observed were of 18 kDa, 44 kDa, 60-62 kDa, 90 kDa, and a doublet of 3840 kDa. Differentiation of monocytes to macrophages by in vitro cultivation was accompanied by a selective decrease in the 60-62 kDa protein. Cultivation of the cells in the presence of the macrophageactivating cytokines interferon-y (IFN-y) and tumor necrosis factor-γ (TNF-γ), prevented the decrease in the expression of the 60—62 kDa myristoylated protein. The effect of cytokines was observed when monocytes were treated with IFN-γ or TNF-α for 24 or 48 h and protein myristoylation analyzed at day four of culture. Maintenance of monocytes in culture for up to nine days in the presence of cytokines prevented the decrease in the expression of the 60-62 kDa myristoylated protein. IFN-γ had additional effects on myristoylation of macrophage proteins. Treatment of monocytes with IFN-y for a few hours caused the induction of a 66 kDa protein. Induction of this myristoylated protein by IFN-γ was time-dependent and peaked at six hours. Analysis of the subcellular distribution of the 66 kDa protein induced by IFN-γ showed that, analogously to other myristoylated proteins, most of it was associated with cell membranes. Initial attempts to identify the myristoylated proteins in human mononuclear pha-gocytyes showed that p60src is one of the myristoylated proteins which is down-modulated during differentiation of monocytes to macrophages, and that cultivation of monocytes in the presence of IFNγ and TNF-α prevents this phenomenon.
Temporal control of differentiation by the cell nucleus: evidence for a species-specific nuclear suppression of enzymes at the early generative phase in Acetabularia
Authors: Bannwarth Horst , Groot Gon J. De 
Published: April 2021

ABSTRACT
The role of the cell nucleus in controling steps of differentiation has been revealed by comparing nucleate and anucleate cells of Acetabularia (Berger et al. 1987; Bonotto, 1988). In all cases investigated so far, events related to the generative phase of the life cycle are expressed earlier in anucleate cells. Consequently, the existence of a nuclear suppressor delaying the onset of generative functions in the intact cell has been postulated (Bannwarth and Schweiger, 1983). In order to determine whether this nuclear action is species-specific, the nucleus of the species Acetabularia acetabulum was replaced by a nucleus of the species Acetabularia crenulata. The foreign nucleus did not suppress the increase in dCMP deaminase (EC 3.5.4.12) enzyme activity. This result indicates that the suppression by the nucleus is species-specific.
Normal Chlamydomonas nuclear gene structure on linkage group xix
Authors: Schloss Jeffery A. , Croom Henrietta Brown 
Published: April 2021

ABSTRACT
The unusual Chlamydomonas linkage group XIX - called the uni linkage group for the uni mutants that lack one of the paired flagellae of wild-type cells - has been reported to be physically located exclusively at the basal bodies. To learn whether the structure of genes on this linkage group differs from the structure of nuclear genes in this organism, we determined the primary structure of a gene that maps to linkage group XIX. This analysis reveals the presence of nine intervening sequences; the nucleotides at exon/intron boundaries conform with nuclear gene intron junction sequences. Also typical for C. reinhardtii nuclear genes are the position and sequence of the putative polyadenylation signal. These findings suggest that transcripts from linkage group XIX are likely to be processed in the nucleus. The open reading frame, which displays weak but easily detected Chlamydomonas codon bias, potentially encodes a protein similar to a membrane anchor for cytoskeletal proteins. The observation that expression of this gene is regulated during interphase and in gametes is not consistent with the hypothesis that linkage group XIX may be expressed only during mitotic and meiotic processes.
Deoxyguanosine enhances the cytotoxicity of the topoisomerase i inhibitor camptothecin by reducing the repair of double-strand breaks induced in replicating dna
Authors: Squires Shoshana , Ryan Anderson J. , Strutt Helen L. , Smith Paul J. , Johnson Robert T. 
Published: April 2021

ABSTRACT
Deoxyguanosine (dG) enhances the S phase cytotoxicity of camptothecin (CPT), a topoisomerase I (topo I) inhibitor, but by contrast does not affect the toxicity of VM26, a topoisomerase II inhibitor. The 80% survival of S phase human fibroblasts after a 60 min exposure to 0.2 pM CPT is reduced by half in the presence of 25 μM dG. Gi cells are resistant to CPT toxicity, though the levels of the single-strand DNA breaks induced by the drug are similar in Gi and S phase cells. Higher concentrations of dG retard the recovery of RNA and DNA synthesis and inhibit recovery from the S-G2 cycle block after CPT removal. At 100 μM dG the number of CPT-induced protein-linked single-strand DNA breaks is almost doubled, suggestive of a direct effect of dG on the cellular activity of topo I. In the presence or absence of dG, single-strand breaks disappear within minutes of the removal of CPT.
We found that the inhibition of topo I by CPT induces the formation of double as well as singlestrand breaks in the chromosomal DNA. Previously we have shown, using a pulse-field gel electrophoresis technique, that the double-strand breaks (DSBs) are generated predominantly at sites of replication and not in the bulk DNA. A number of these DSBs are long-lived. The present study shows that dG affects the repair of these DSBs in a dose-dependent manner, and that a higher proportion of the initial lesions induced in nascent DNA remain 24 h after removal of CPT. We suggest that the long-lived double-strand breaks, formed in replicating DNA at the time of CPT exposure, are the lethal drug-induced lesions, which explains both the selective cytotoxicity of CPT towards S phase cells and the enhancement of CPT cytotoxicity by dG.
Analysis of p53 expression in human tumours: an antibody raised against human p53 expressed in Escherichia coli
Authors: Midgley C. A. , Fisher C. J. , Bártek J. , Vojtések B. , Lane D. , Barnes D. M. 
Published: April 2021

ABSTRACT
A cDNA encoding the complete normal human p53 protein was expressed in Escherichia coli using an expression system based on the bacteriophage T7 promoter. The cDNA was adapted so that the full-length protein was produced without fusion to any other sequence. Large amounts of the protein were isolated and the purified protein used to produce very high titre polyclonal antibodies to p53. These new antibodies permit the sensitive detection of p53 and p53 complexes in ELISA and immunoblotting assays. Most importantly, they also permit the detection of p53 in archival tumour material that has been conventionally fixed in formalin and embedded in paraffin wax. Using this reagent we have found that aberrant expression of p53 is a frequent feature of human breast cancer. We are able to recognise six different classes of p53 expression pattern that may be of help in the subclassification of breast tumours.
Enhanced extracellular matrix production and mineralization by osteoblasts cultured on titanium surfaces in vitro
Authors: Groessner-Schreiber Birte , Tuan Rocky S. 
Published: April 2021

ABSTRACT
Long-term stability of orthopaedic and dental implants depends on the integration of the artificial material into the surrounding bone tissue. The physical and chemical properties of implants, including those made of metals such as titanium, are thought to influence osseointeg-ration. Despite the known importance of this interface, little is known about the factors that promote its formation. In this study, chick embryonic calvarial osteoblasts were cultured in vitro on smooth, rough-textured and porous-coated titanium surfaces and examined for morphology, biosynthesis of extracellular matrix and mineralization as a function of culture time. Scanning electron microscopy revealed that osteoblasts adhered securely to the titanium surface and frequently bridged the uneven surface by means of cellular processes. The osteoblast phenotype was retained in the cell cultures on titanium. In addition, the synthesis of extracellular matrix and subsequent mineralization were both substantially enhanced in the cultures on rough-textured and porous-coated titanium. These results strongly suggest that porous or rough titanium implant surfaces may act like “natural” substrata to permit microscopic tissue/cell ingrowth to improve clinical implant fixation.
Distribution of the myosin i-like ninaC proteins in the Drosophila retina and ultrastructural analysis of mutant phenotypes
Authors: Hicks Jennifer L. , Williams David S. 
Published: April 2021

ABSTRACT
The Drosophila ninaC gene encodes for two head-specific proteins of 132 kDa and 174 kDa. Their predicted amino acid sequences indicate that they may have myosin I and kinase properties. We have: (1) determined the cellular and subcellular distributions of the ninaC proteins in the Drosophila retina by electron microscopic immunocytochemistry with an antibody specific for epitopes shared by both proteins; (2) characterized the ultrastructure of the mutant phenotype. The proteins were detected only in the photoreceptor cells, but were detected in all classes of the compound eye photoreceptors. Within the photoreceptors, they were found in the rhabdomeral microvilli and the cytoplasm adjacent to the rhabdo-meres. This distribution coincides with that shown previously for actin filaments. Immunolabelling of tissue from the ninaC P221 mutant, which lacks the 174 kDa protein, and two mutants whose rhabdomeres degenerate, suggests that the 132 kDa protein is present primarily in the cytoplasm adjacent to the rhabdomeres, and that the 174 kDa protein is concentrated in the rhabdomeres. Our ultrastructural analysis showed that the axial cytoskeleton of the rhabdomeral microvilli (which contains filamentous actin) was absent in both the null and P221 mutants. In the photoreceptor cell cytoplasm, the number of multivesicular bodies in the null mutant, but not the P221 mutant, was 3-fold greater in comparison with wild-type. Together, the immunolabelling results and the ultrastructural observations of the mutant phenotypes are consistent with the 174 kDa protein functioning as a component of the microvillar cytoskeleton (perhaps in an analogous manner to the 110 kDa brush border myosin I), and the 132 kDa protein playing a role in the adjacent cytoplasm by effecting some aspect of photoreceptor membrane turnover.
The role of primer recognition proteins in DNA replication: inhibition of cellular proliferation by antisense oligodeoxyribonucleotides
Authors: Kumble Krishnanand D. , Iversen Patrick L. , Vishwanatha Jamboor K. 
Published: April 2021

ABSTRACT
Primer recognition proteins (PRP) are cofactors for DNA polymerase α and may have a role in laggingstrand DNA replication. PRP is composed of two subunits, which we have previously identified as the protein-tyrosine kinase substrate annexin U and phosphoglycerate kinase (PGK). In this study, we have examined the physiological involvement of these proteins in DNA synthesis and cell proliferation. When exponentially growing human HeLa cells are exposed to antisense phosphorothioate oligodeoxynucleotides to annexin H, ongoing DNA synthesis is reduced. The extent of reduction with antisense oligodeoxynucleotide to PGK was much less than with the antisense annexin n oligodeoxy nucleotide. Reductions in the labeling and mitotic indices of HeLa cell cultures are seen after exposure to antisense oligodeoxynucleotides. Flow cytometric analyses indicate that progression from S phase to G2 phase of the cycle is retarded by exposure of cells to the antisense oligodeoxynucleotides. Corresponding sense oligodeoxynucleotides have no inhibitory effects on these parameters. The new synthesis of annexin H and PGK is specifically reduced in the presence of antisense oligodeoxynucleotides, indicating that the complex of newly synthesized annexin H and PGK may participate in PRP function. These experiments indicate that annexin H and PGK may have a physiological role in DNA synthesis and cell cycle progression, and represent the first physiological role for annexin H monomer in cells.
The increase in intracellular ph associated with Xenopus egg activation is a ca2+-dependent wave
Authors: Grandin Nathalie , Charbonneau Michel 
Published: April 2021

ABSTRACT
In Xenopus eggs, the transient increase in intracellular free calcium ([Ca2+]i), or Ca2+ transient, which occurs 1–3 min after egg activation, is likely to be partly responsible for the release of the cell cycle blockade. In the present study, we have used microinjection of BAPTA or EGTA, two potent chelators of Ca2+, to buffer [Ca2+] i at various steps during Xenopus egg activation and evaluate the impact on some of the associated events. Microinjection of either one of the Ca2+ chelators into unactivated eggs prevented egg activation without, however, lowering [Ca2+]i, suggesting that only physiological [Ca2+]i changes, but not [Ca2+]1 levels, were affected by the Ca2+ buffer. When BAPTA was microinjected around the time of occurrence of the Ca2+ transient, the egg activation-associated increase in intracellular pH (pH0 was clearly delayed. That delay was not due to a general slowing down of the cell cycle, since under the same conditions of microinjection of BAPTA the kinetics of MPF (a universal M-phase promoting factor) inactivation were unaffected. These results represent the first indication that the Ca2+ transient participates in determining the time of initiation of the pH1 increase during Xenopus egg activation. The present results also demonstrate that the egg activation-associated pHi changes (a slight, transient decrease in pH, followed by a permanent increase in pH|) proceed as a wave propagating from the site of triggering of egg activation. Experiments of local microinjection of BAPTA support the view that the pH wave is a consequence of the Ca2+ wave, which it follows closely.
Free ca2+ gradient in growing pollen tubes oflilium
Authors: Miller Deborah D. , Callaham Dale A. , Gross David I. , Hepler Peter K. 
Published: April 2021

ABSTRACTFluorescence ratiometric imaging of Lilium pollen tubes loaded with the Ca2+ indicator Fura-2 dextran has revealed a distinct elevation of free intracellular calcium ion concentration ([Ca2+]i) at the extreme tip of actively growing Lilium pollen tubes that declines to a uniform basal level of ∼170 nM throughout the length of the tube. The calcium gradient occurs within the first 10–20 μM proximal to the tip. Experimental inhibition of tip growth, usually achieved through the injection of the Ca2+ buffer 5,5’-dibromo BAPTA, results in the loss of the [Ca2+]igradient. Occasionally these inhibited cells reinitiate growth, and when they do so ratio imaging reveals that the tip gradient of free [Ca2+Ji re-emerges. The results presented here are very different from those previously published by revealing the presence of the [Ca2+]i gradient that is restricted to the 10–20 μM adjacent to the tube tip. Further, these experiments demonstrate a strict correlation between the presence of a [Ca2+Ji gradient, and tip growth in Lilium pollen tubes.
Disruption of synchrony between parasite growth and host cell division is a determinant of differentiation to the merozoite in Theileria annulata
Authors: Shiels Brian , Kinnaird Jane , Mckellar Susan , Dickson Joanne , Miled Leila Ben , Melrose Ron , Brown Duncan , Tait Andy 
Published: April 2021

ABSTRACT
The multinucleated macroschizont stage of the proto-zoon Theileria annulata is an intracellular parasite of bovine leukocytes. The parasite induces the host cell to proliferate, and divides in synchrony with the immortalised host cell. Differentiation to the next stage occurs within the host cell culminating in the release of merozoites and destruction of the leukocyte. In this study clones of Theileria annulata macroschizont-infected cell lines were isolated by limiting dilution and tested for differentiation to the merozoite stage (mero-gony). Two cloned cell lines underwent differentiation with enhanced efficiency, while two others were of lower efficiency. Quantification was carried out using monoclonal antibodies, which showed that over 90% of the cells in an enhanced cloned cell line could be induced to differentiate. By carrying out induction at 41°C for limited periods of time followed by culture at 37°C evidence was obtained that differentiation to the merozoite is a two-step process: a preliminary reversible phase, followed by a second irreversible phase of differentiation. Analysis of the nuclear number of the macroschizont and the growth rate of the cloned cell lines showed that the ability to differentiate was associated with an increase in nuclear number (size) of the macroschizont, generated by a disruption in the synchrony between parasite growth and host cell division. We believe that these results reveal a relationship between a reduction in parasite division and differentiation, and that there are similarities between stage differentiation in parasites and cellular differentiation in higher eukaryotes.
Effects of vinblastine, podophyllotoxin and nocodazole on mitotic spindles : Implications for the role of microtubule dynamics in mitosis
Authors: Ann Jordan Mary , Thrower Douglas , Wilson Leslie 
Published: April 2021

ABSTRACT
Inhibition of mitosis by many drugs that bind to tubulin has been attributed to depolymerization of microtubules. However, we found previously that low concentrations of vinblastine and vincristine blocked mitosis in HeLa cells with little or no depolymerization of spindle microtubules, and spindles appeared morphologically normal or nearly normal. In the present study, we characterized the effects of vinblastine, podophyllotoxin and nocodazole over broad concentration ranges on mitotic spindle organization in HeLa cells. These three drugs are known to affect the dynamics of microtubule polymerization in vitro and to depolymerize microtubules in cells. We wanted to probe further whether mitotic inhibition by these drugs is brought about by a more subtle effect on the microtubules than net microtubule depolymerization. We compared the effects of vinblastine, podophyllotoxin and nocodazole on the organization of spindle microtubules, chromosomes and centrosomes, and on the total mass of microtubules. Spindle organization was examined by immunofluorescence microscopy, and microtubule polymer mass was assayed on isolated cytoskeletons by a quantitative enzyme-linked immunoadsorbence assay for tubulin. As the drug concentration was increased, the organization of mitotic spindles changed in the same way with all three drugs. The changes were associated with mitotic arrest, but were not necessarily accompanied by net microtubule depolymerization. With podophyllotoxin, mitotic arrest was accompanied by microtubule depolymerization. In contrast, with vinblastine and nocodazole, mitotic arrest occurred in the presence of a full complement of spindle microtubules. All three drugs induced a nearly identical rearrangement of spindle microtubules, an increasingly aberrant organization of metaphase chromosomes, and fragmentation of centrosomes. The data suggest that these anti-mitotic drugs block mitosis primarily by inhibiting the dynamics of spindle microtubules rather than by simply depolymerizing the microtubules.
Cell cycle events in the green alga Chlamydomonas eugametos and their control by environmental factors
Authors: Zachleder V. , Ende H. Van Den 
Published: April 2021

ABSTRACT
A procedure for routine synchronization of large amounts of the unicellular green alga Chlamydomonas eugametos in liquid culture by alternating light and dark periods is described. The synchronized populations were grown at various light intensities and temperatures. The effect of these variables on the lengths of parts of the cell cycle and the number of daughter cells per cell division was followed. The cell cycle of C. eugametos started with a period in which the cells increased in size only (precommitment period). The length of this period was dependent on both the light intensity and the temperature. At the end of this period, a key point of the cell cycle (called commitment point) was attained. From this point, the cell were committed to divide and cell reproduction was triggered. The following period (postcommitment period), during which daughter cells were formed, could be traversed without supply of external energy, and without further growth of the cells. However, if sufficient energy was supplied during this period, the cells were able to attain more commitment points, leading to a higher number of daughter cells. The postcommitment period was fairly constant within a certain range of light intensity. At light intensities leading to more commitment points, however, this period was prolonged. No evidence was found for circadian rhythms or endogenous factors of “Zeitgeber” type playing a role in the control of growth and reproductive sequences in the cell cycle of C. eugametos.
Autophagic vacuoles rapidly fuse with pre-existing lysosomes in cultured hepatocytes
Authors: Lawrence B. Paige , Brown William J. 
Published: April 2021

ABSTRACT
Autophagic vacuoles (A Vs) arise when membranes of the ER sequester parts of the cytoplasm, forming a new, double-membraned vacuole, to which lysosomal enzymes are then delivered. To investigate the mechanism of lysosomal enzyme delivery to nascent AVs, amino acid (AA) starvation and glucagon treatment were used to induce autophagy in a cultured cell system using rat hepatocytes (Fu5C8 cells). The induction of autophagy was assayed using biochemical, morphometric and immunocytochemical techniques. In these cells, AA starvation resulted in a fivefold increase in total cellular proteolysis, and sixfold and 4.5-fold increases in the volume and surface densities of AVs, respectively. Using an antibody against the mannose 6-phosphate receptor (MPR) and two sizes of colloidal gold to label separately and track the endosomal and lysosomal compartments, the time course of endosomal and lysosomal fusion with AVs was analyzed in detail. On the basis of these experiments, we found that AVs rapidly fuse with preexisting lysosomes, but seldom with a prelysosomal compartment (PLC). Using immunoperoxidase, staining for the MPR was infrequently observed in association with any AVs. However, at early times following the induction of autophagy (&lt;2 h), many autophagic vacuoles stained positively for the lysosomal enzyme cathepsin D. Consistent with these results, treatment of cells with tunicamycin had no effect on autophagy-induced proteolysis. We conclude that lysosomal enzyme delivery to nascent AVs occurs primarily by the fusion of pre-existing mature lysosomes, with a much smaller contribution by MPRs or the PLC.