bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effects of activators (SC-9 and OAG) and inhibitors (staurosporine and H-7) of protein kinase C on the proliferation of mouse epidermal melanoblasts in serum-free culture
Authors: Hirobe Tomohisa 
Published: April 2021

ABSTRACT
Mouse epidermal melanoblasts preferentially proliferated from disaggregated epidermal cell suspensions derived from newborn mouse skin in a serum-free melanoblast proliferation medium containing dibutyryl adenosine 3′:5′cyclic monophosphate and basic fibroblast growth factor. After 12 days, almost all of the keratinocytes died and pure cultures of melanoblasts (∼80%) and melanocytes (∼20%) could be obtained. No further proliferation of melanoblasts was observed in the melanoblast proliferation medium. In order to clarify the role of protein kinase C, which is important for the regulation of cellular proliferation, activators or inhibitors of protein kinase C were added to the culture of the quiescent melanoblasts at 12 days. The proliferation of melanoblasts was induced by an activator of protein kinase C, N-(6-phenylhexyl)-5-chloro-1-naphthalene-sulfonamide or 1-oleoyl-2-acetyl-glycerol. It was also induced by an inhibitor of protein kinase C, staurosporine or 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine. However, the melanoblasts failed to proliferate in the melanoblast proliferation medium supplemented with both the activator and the inhibitor. These results suggest that the proliferation of mouse epidermal melanoblasts in culture is regulated by activating or inhibiting the activity of protein kinase C.
Characterization of cadherin-4 and cadherin-5 reveals new aspects of cadherins
Authors: Tanihara Hidenobu , Kido Mitsuru , Obata Shuichi , Heimark Ronald L. , Davidson Mari , John Tom St , Suzuki Shintaro 
Published: April 2021

ABSTRACT
Several properties of cadherin-4 and cadherin-5 were char- acterized by using the cDNA transfection approach. The proteins of both cadherins had a relative molecular mass of about 130 kDa and were present at the cell periphery, especially at cell-cell contact sites. These cadherins were easily digested with trypsin, and Ca2+ protected cadherin- 4, but not cadherin-5, from the digestion. In immunopre- cipitation, cadherin-4 co-precipitated with two major proteins of 105 kDa and 95 kDa, respectively. The 105 kDa and the 95 kDa proteins are likely to correspond to α- and β-catenins. Cadherin-5 co-precipitated with only one major protein of 95 kDa, but seems to associate with the 105 kDa protein. On the other hand, plakoglobin or γ-catenin did not co-precipitate well with either cadherin-4 or cadherin- 5 in immunoprecipitation, but plakoglobin also appears to associate weakly with these cadherins. Cadherin-4 trans- fectants aggregated within 30 minutes in a cell aggregation assay, but cadherin-5 transfectants did not aggregate under the same conditions. Furthermore, the transfectants of chimeric cadherin-4 with cadherin-5 cytoplasmic domain showed cell aggregation activity comparable to that of wild- type cadherin-4 transfectants, whereas the transfectants of chimeric cadherin-5 with cadherin-4 cytoplasmic domain did not show appreciable cell aggregation, suggesting that the extracellular domains of cadherins, in conjunction with their cytoplasmic domains, play an important role in cell aggregation activity. These results show that cadherin-4 is very similar to the classical cadherins, whereas cadherin-5 is functionally as well as structurally distinct from classical cadherins.
Sites in human nuclei where damage induced by ultraviolet light is repaired: localization relative to transcription sites and concentrations of proliferating cell nuclear antigen and the tumour suppressor protein, p53
Authors: Jackson Dean A. , Hassan A. Bass , Errington Rachel J. , Cook Peter R. 
Published: April 2021

ABSTRACT
The repair of damage induced in DNA by ultraviolet light involves excision of the damaged sequence and synthesis of new DNA to repair the gap. Sites of such repair synthesis were visualized by incubating permeabilized HeLa or MRC-5 cells with the DNA precursor, biotin-dUTP, in a physiological buffer; then incorporated biotin was immunolabelled with fluorescent antibodies. Repair did not take place at sites that reflected the DNA distribution; rather, sites were focally concentrated in a complex pattern. This pattern changed with time; initially intense repair took place at transcriptionally active sites but when transcription became inhibited it continued at sites with little transcription. Repair synthesis in vitro also occurred in the absence of transcription. Repair sites generally contained a high concentration of proliferating cell nuclear antigen but not the tumour-suppressor protein, p53.
Modulation of α-actinin levels affects cell motility and confers tumorigenicity on 3T3 cells
Authors: Glück Ursula , Ben-Ze’ev Avri 
Published: April 2021

ABSTRACT
α-Actinin is an abundant actin crosslinking protein, also localized at adherens type junctions. In adhesion plaques, α-actinin can link the actin filaments to integrin via vinculin and talin, or directly by binding to the cytoplasmic domain of β1-integrin. The expression of α-actinin is rapidly elevated in growth-activated quiescent cells, and is reduced in SV40-transformed 3T3 cells and various differentiating cell types (reviewed by Glück, U., Kwiatkowski, D. J. and Ben-Ze’ev, A. Proc. Nat. Acad. Sci. USA 90, 383-387, 1993). To study the effect of changes in α-actinin levels on cell behavior, α-actinin expression was elevated in 3T3 cells by transfection with a full-length human nonmuscle α-actinin cDNA. To suppress α-actinin levels, 3T3 cells were transfected with an antisense α-actinin cDNA construct. Cells overexpressing α-actinin by 40-60% displayed a significant reduction in cell motility, as demonstrated by their slower locomotion into an artificial wound, and by forming shorter phagokinetic tracks on colloidal gold-coated substrata. 3T3 cells in which the expression of α-actinin was reduced to 25-60% of control levels, after antisense α-actinin transfection, had an increased cell motility. Moreover, such α-actinin-deficient 3T3 cells formed tumors upon injection into nude mice. The results demonstrate that modulations in α-actinin expression can affect, in a major way, the motile and tumorigenic properties of cells, and support the view that decreased α-actinin expression could be a common regulatory pathway to malignant transformation of 3T3 cells.
The mode of anchorage to the cell surface determines both the function and the membrane location of Thy-1 glycoprotein
Authors: Tiveron M.-C. , Nosten-Bertrand M. , Jani H. , Garnett D. , Hirst E. M. A. , Grosveld F. , Morris R. J. 
Published: April 2021

ABSTRACT
The surface glycoprotein, Thy-1, when expressed by transfection in NG115/401L neural cells, inhibits their neurite outgrowth over astrocytes. We have investigated the role of the glycosylphosphatidylinositol anchor of Thy-1 in this inhibition. Hybrid molecules, in which the lipid anchor was replaced by polypeptide transmembrane domains, were expressed by transfection. Lines expressing Thy-1 with the transmembrane and full cytoplasmic domains of NCAM-140, or with the transmembrane and truncated cytoplasmic domain of CD8, were not inhibited in their ability to extend neurites over astrocytes. Truncation of the cyto-plasmic domain of NCAM-140 to just two amino acids, however, produced a transmembrane form of Thy-1 that, when expressed at high levels, inhibited neurite outgrowth. All forms of Thy-1 were concentrated in clusters that occurred primarily on fine filopodia. In double transfectants expressing normal Thy-1 and Thy-1 with the full NCAM cytoplasmic tail, the clusters of each form were separate, with no instances of the transmembrane form being found within the clusters of lipid-anchored Thy-1. Thy-1 with the two-amino-acid cytoplasmic domain of NCAM also occurred in clusters separate from those occupied by lipid-anchored Thy-1, but substantial ‘invasion’ of the clusters of normal Thy-1 by this trans-membrane construct occurred. We suggest that the ability of this hybrid protein to enter the lipid-anchored clusters enables it to activate the signalling pathways that normal Thy-1 uses. Thus the membrane anchor, in targetting Thy-1 to different microdomains on the cell surface, determines its ability to inhibit neurite outgrowth on astrocytes.
Mitotic arrest with anti-microtubule agents or okadaic acid is associated with increased glycoprotein terminal GlcNAc’s
Authors: Chou Chih-Fong , Omary M. Bishr 
Published: April 2021

ABSTRACT
The two major intermediate filament glycoproteins in human simple epithelia are keratins 8 and 18 (K8/18). A dramatic increase in terminal N-acetylglucosamine (GlcNAc) residues in K8/18 was previously noted after arresting cells in G2/M using anti-microtubule agents. Here we use in vitro galactosylation to show that increased terminal GlcNAc’s is a general phenomenon that occurs in glycoproteins isolated from nuclear and plasma membrane fractions after cells are arrested in mitosis using colcemid, nocodazole, or okadaic acid. All three agents also resulted in a hyperphosphorylated form of K8 as determined by phosphatase treatment and tryptic phosphopeptide mapping. The altered glycosylation was found to be independent of microtubule disassembly, and was not directly related to the G2/M phase of the cell cycle after aphidicolin synchronization. Staurosporine (1 μM) inhibited K8/18 phosphorylation in okadaic acid- or nocodazole-treated cells, and inhibited the increase in K8/18 glycosylation without inhibiting the increase in terminal GlcNAc’s of membrane-associated glycoproteins. In contrast, brefeldin A resulted in a dramatic increase in terminal GlcNAc’s of membrane-associated but not intermediate filament proteins. Golgi complex-related staining using anti-β-COP antibody showed significant fragmentation under conditions associated with altered membrane protein glycosylation. Our results suggest that Golgi disruption may be involved in the observed increase in terminal GlcNAc’s of membrane but not intermediate filament glycoproteins. The mechanism of increased glycoprotein terminal GlcNAc’s in association with mitotic arrest appears to be distinct for intermediate filaments and membrane-associated proteins, and in the case of intermediate filament proteins, phosphorylation may play an important role. Some of the effects of agents that induce mitotic arrest may be mediated by glycosylation changes.
A homologue of the human regulator of mitotic spindle assembly protein (RMSA-1) is present in crane fly and is associated with meiotic chromosomes
Authors: Yeo Jing-Ping , Forer Arthur , Toh Ban-Hock 
Published: April 2021

ABSTRACT
In a previous study, we have shown that a newly identified chromosomal protein, RMSA-1 (Regulator of Mitotic Spindle Assembly-1), identified and cloned using a human autoimmune serum, is essential for mitotic spindle assembly; we proposed that RMSA-1 was a previously unknown physiological substrate for cdc 2 kinase. In the present study, we show that this protein is present in crane fly and is associated with the chromosomes of spermatocytes.
A 31 kDa molecule in extracts from crane-fly nuclei, isolated from larvae, pupae and adults, reacts with affinity-purified anti-RMSA-1 autoantibody, shown by immunoblotting. The autoantibody reacts, as shown by immunofluorescence, with crane-fly spermatocyte chromosomes in prophase through anaphase of both meiosis-1 and meiosis-II but does not react with preprophase or telophase nuclei or with spermatid nuclei. In all meiotic stages, the crane-fly sex chromosomes stain more intensely than the autosomes. We conclude that, since RMSA-1 is present in insect and mammalian cells, it is conserved across a variety of animal species. Further, since RMSA-1 binds to chromosomes in meiotic cells, it also may be essential for assembly of the meiotic spindle.
Molecular phylogeny of the kinesin family of microtubule motor proteins
Authors: Goodson Holly V. , Kang Sang Joon , Endow Sharyn A. 
Published: April 2021

ABSTRACT
The rapidly expanding kinesin family of microtubule motor proteins includes proteins that are involved in diverse microtubule-based functions in the cell. Phylogenetic analysis of the motor regions of the kinesin proteins reveals at least five clearly defined groups that are likely to identify kinesins with different roles in basic cellular processes. Two of the groups are consistent with overall sequence similarity, while two groups contain proteins that are related in overall structure or function but show no significant sequence similarity outside the motor domain. One of these groups consists only of kinesin proteins with predicted C-terminal motor domains; another includes only kinesins required for mitotic spindle bipolarity. Drosophila Nod, presently an ungrouped protein, may represent a class of kinesins that, like the myosin I proteins, function as monomers. The analysis indicates that many types of kinesin proteins exist in eukaryotic organisms. At least two of the five groups identified in this analysis are expected to be present in most, or all, eukaryotes.
Movement of membrane tubules along microtubules in vitro: evidence for specialised sites of motor attachment
Authors: Allan Viki , Vale Ronald 
Published: April 2021

ABSTRACT
We have studied the microtubule-dependent formation of tubular membrane networks in vitro, using a heterologous system composed of Xenopus egg cytosol combined with rat liver membrane fractions enriched in either Golgi stacks or rough endoplasmic reticulum. The first step in membrane network construction involves the extension of membrane tubules along microtubules by the action of microtubule-based motor proteins. We have observed for both membrane fractions that 80-95% of moving tubule tips possess a distinct globular domain. These structures do not form simply as a consequence of motor protein activity, but are stable domains that appear to be enriched in active microtubule motors. Negative stain electron microscopy reveals that the motile globular domains associated with the RER networks are generally smaller than those observed in networks derived from a crude Golgi stack fraction. The globular domains from the Golgi fraction are often packed with very low density lipoprotein particles (the major secretory product of hepatocytes) and albumin, which suggests that motor proteins may be specifically enriched in organelle regions where proteins for export are accumulated. These data raise the possibility that the concentration of active motor proteins into specialised membrane domains may be an important feature of the secretory pathway.
Actin microfilaments are associated with the migrating nucleus and the cell cortex in the green alga Micrasterias Studies on living cells
Authors: Meindl Ursula , Zhang Dahong , Hepler Peter K. 
Published: April 2021

ABSTRACT
Rhodamine-phalloidin or FITC-phalloidin has been injected in small amounts into living, developing cells of Micrasterias denticulata and the stained microfilaments visualized by confocal laser scanning microscopy. The results reveal that two different actin filament systems are present in a growing cell: a cortical actin network that covers the inner surface of the cell and is extended far into the tips of the lobes in both the growing and the non-growing semicell; it is also associated with the surface of the chloroplast. The second actin system ensheathes the nucleus at the isthmus-facing side during nuclear migration. Its arrangement corresponds to that of the microtubule system that has been described in earlier electron microscopic investigations. The spatial correspondence between the distribution of actin filaments and microtubules suggests a cooperation between both cytoskeleton elements in generating the motive force for nuclear migration. The function of the cortical actin network is not yet clear. It may be involved in processes like transport and fusion of secretory vesicles and may also function in shaping and anchoring the chloroplast.
Dephosphorylation of the largest neurofilament subunit protein influences the structure of crossbridges in reassembled neurofilaments
Authors: Gotow Takahiro , Tanaka Toshihisa , Nakamura Yu , Takeda Masatoshi 
Published: April 2021

ABSTRACT
Phosphorylation-dependent change in electrophoretic mobility is the most unique characteristic of NF-H, the largest molecular mass subunit of the neurofilament. We dephosphorylated NF-H using Escherichia coli alkaline phosphatase, then reassembled it into neurofilaments with NF-M and NF-L, and into NF-H filaments with NF-H alone. We compared these dephosphorylated filaments with control: projections by low-angle rotary-shadow, crossbridges by quick-freeze deep-etch, and core filament packing density by thin-section electron microscopy. Projections in the dephosphorylated filaments were basically similar in structure to those in control, although there was a tendency for them to be wider and less dense, especially in NF-H filaments. Dephosphorylated filaments were still able to form crossbridges between core filaments, but their crossbridges were significantly wider, less dense, more branched and more irregular than crossbridges in control, and core filaments were more densely packed. These structural differences may be brought about by the removal of phosphate groups from NF-H tail and consequent reduction of electrostatic repulsion between adjacent cross-bridges extending from the same core filament. The results indicate that phosphorylation of NF-H is necessary for forming well developed crossbridges, straight and at constant intervals, like those of in vivo axonal neurofilaments.
Function of type I and type II keratin head domains: their role in dimer, tetramer and filament formation
Authors: Hatzfeld Mechthild , Burba Matthias 
Published: April 2021

ABSTRACT
To examine the role of the keratin head region and its subdomains in filament assembly we constructed several deletion mutants of type I and type II keratins and analysed their in vitro IF forming capacity. The ΔK8 (1-74) and 6KIS (1-56) mutants formed only soluble oligomers, predominantly tetramers with their heterotypic partners. KS mutants that retained either the entire ΔK8 (1-64)) or nearly the entire ΔK8 (1-66)) Hl subdomain formed some short and irregular IF-like structures with K18. However, filaments never reached the normal length and more protofilamentous material was observed. Analysis of the soluble complexes in 2 M guanidine-HCI indicated that tetramer formation was impaired in the truncated molecules. The length of the deletion correlated with the degree of tetramer destabilization. These results suggest that the head domain - specifically the Hl subdomain of type II keratins - plays a direct role in IF assembly. Its functions include a stabilization of the tetramer molecule, suggesting a role in directing the alignment of dimers as well as in elongation.
We also analysed whether both head domains are required or if either type I or type II head domains alone are sufficient for IF formation. Hybrid molecules carrying their partner keratins head domains (K1S (8 head) and KS (18 head)) were combined with their wild-type partners and tested for IF-forming ability. Both combinations formed filaments distinct from normal IF. The effect of the ‘replaced’ head domains was not compensated when both hybrid molecules were combined.
Taken together, the results indicate that complete removal of the head domains of either KS or K 18 arrested IF assembly at the state of soluble oligomers. Replacement of the head domains by head domains of the complementary partner partly compensated for the effect. However, regular IF formation could not take place when either the head domain was missing or it was replaced by the partner’s keratin head.
The role of transcription factors, chromatin structure and DNA replication in 5 S RNA gene regulation
Authors: Wolffe Alan P. 
Published: April 2021

ABSTRACT
Differential expression of the oocyte and somatic 5 S RNA genes during Xenopus development can be explained by changes in transcription factor and histone interactions with the two types of gene. Both factors and histones bind 5 S RNA genes with specificity. Protein-protein interactions determine the stability of potentially transcriptionally active or repressed nucleoprotein complexes. A decline in transcription factor abundance, differential binding of transcription factors to oocyte and somatic 5 S genes, and increased competition with the histones for association with DNA during early embryogenesis, can account for the developmental decision to selectively repress the oocyte genes, while retaining the somatic genes in the transcriptionally active state.
The 5 S ribosomal genes of Xenopus are perhaps the simplest eukaryotic genes to show regulated expression during development. A large multigene family (oocyte 5 S DNA) is transcriptionally active in oocytes but is repressed in somatic cells, whereas a small multigene family (somatic 5 S DNA) is active in both cell types. A potential molecular mechanism to explain the developmental switch that turns off oocyte 5 S DNA transcription has been experimentally reconstructed in vitro and more recently tested in vivo. Central to this mechanism is the specific association of both transcription factors and histones with 5 S RNA genes. How the interplay of histones and transcription factors is thought to affect transcription, and how their respective contributions might change during development from an oocyte, to an embryo and eventually to a somatic cell is the focus of this review.
Structural motifs for recognition and adhesion in members of the immunoglobulin superfamily
Authors: Holness Claire L. , Simmons David L. 
Published: April 2021

ABSTRACT
The immunoglobulin superfamily is the most abundant family of cell surface molecules, accounting for 50% of leukocyte surface glycoproteins. This evolutionary success story is thought to be due to the stability of the Ig domain, which is able to resist the harsh proteolytic and oxidative environment of the extracellular world. By mutation and selection, the Ig domain has evolved to serve many different functions including: receptors for growth factors (CSF-1 receptor, PDGF receptor, FGF receptors); receptors for the Fc region of Ig (IgG receptors; CD 16, CD32, CD64; CD89 IgA receptor); and as adhesion molecules, which now seems to be the function of the majority (CD2/CD58, CD28 and CTLA4, which bind to B7 and B70; CD4/class II, CD8/class I, CD31/CD31, CD50 (ICAM-3)/LFA-1, CD54 (ICAM-1)/LFA-1, CD102 (ICAM-2)/LFA-1, CD106 (VCAM)/VLA-4, NCAM/NCAM. L1, MAG, TAG-1, CEA) (Springer, 1990).