bluuebunny/crossref_metadata_2025_split huggingface dataset:
Autoantibodies to a novel cell cycle-regulated protein that accumulates in the nuclear matrix during S phase and is localized in the kinetochores and spindle midzone during mitosis
Authors: Casiano Carlos A. , Landberg Göran , Ochs Robert L. , Tan Eng M. 
Published: April 2021

ABSTRACT
We have employed human autoantibodies to character-ize a novel cell cycle-regulated nuclear protein, provi-sionally designated p330d (doublet polypeptide of 330 kDa). The expression and intracellular distribution of this protein was followed throughout the cell cycle using immunofluorescence microscopy, laser confocal microscopy, immunoelectron microscopy and flow cytometry. p330d was expressed only in proliferating cells and began accumulating in the nucleus during early S phase. The protein reached maximum expression levels during G2/M. In situ extractions with detergent, salt and nucleases failed to abolish the nuclear staining of interphase cells, suggesting a tight binding of p330d to the nuclear matrix during interphase. p330d was con-centrated in the kinetochores during prophase but was relocated to the spindle midzone at the onset of anaphase. By late telophase, it was localized predomi-nantly in the intercellular bridge regions flanking the midbody and disappeared gradually as the daughter cells separated. Immunoblotting analysis showed that the autoimmune sera recognized a doublet of 330 kDa, and affinity-purified antibodies from this doublet repro-duced the fluorescence staining pattern of the whole serum. We propose that p330d is a novel member of the class of ‘chromosomal passenger’ proteins, which are associated transiently with centromeres during early mitosis and are then redistributed to other sites of the mitotic apparatus after the metaphase/anaphase transi-tion. Possible in vivo functions for p330d and related proteins might include roles in centromere/kinetochore maturation and assembly, chromosome segregation, central spindle stabilization and cytokinesis.
Numerical analysis of a comprehensive model of M-phase control in Xenopus oocyte extracts and intact embryos
Authors: Novak Bela , Tyson John J. 
Published: April 2021

ABSTRACT
To contribute to a deeper understanding of M-phase control in eukaryotic cells, we have contructed a model based on the biochemistry of M-phase promoting factor (MPF) in Xenopus oocyte extracts, where there is evi-dence for two positive feedback loops (MPF stimulates its own production by activating Cdc25 and inhibiting Wee1) and a negative feedback loop (MPF stimulates its own destruction by indirectly activating the ubiquitin pathway that degrades its cyclin subunit). To uncover the full dynamical possibilities of the control system, we translate the regulatory network into a set of differential equations and study these equations by graphical techniques and computer simulation. The positive feed-back loops in the model account for thresholds and time lags in cyclin-induced and MPF-induced activation of MPF, and the model can be fitted quantitatively to these experimental observations. The negative feedback loop is consistent with observed time lags in MPF-induced cyclin degradation. Furthermore, our model indicates that there are two possible mechanisms for autonomous oscillations. One is driven by the positive feedback loops, resulting in phosphorylation and abrupt dephosphorylation of the Cdc2 subunit at an inhibitory tyrosine residue. These oscillations are typical of oocyte extracts. The other type is driven by the negative feedback loop, involving rapid cyclin turnover and negligible phosphorylation of the tyrosine residue of Cdc2. The early mitotic cycles of intact embryos exhibit such character-istics. In addition, by assuming that unreplicated DNA interferes with M-phase initiation by activating the phosphatases that oppose MPF in the positive feedback loops, we can simulate the effect of addition of sperm nuclei to oocyte extracts, and the lengthening of cycle times at the mid-blastula transition of intact embryos.
Abnormal cytoskeletal and chromosome distribution in po, ms4 and ms6; mutant alleles of polymitotic that disrupt the cell cycle progression from meiosis to mitosis in maize
Authors: Liu Qinqin , Golubovskaya Inna , Cande W. Zacheus 
Published: April 2021

ABSTRACT
The maize cell cycle regulation mutant polymitotic (po) progresses through abnormal cell cycles, characterized by premature cell divisions without chromosome dupli-cation of the daughter cells produced by meiosis during microsporogenesis and macrosporogenesis. There are three recessive alleles of the Po gene; po, ms4, and ms6. A new method of permeabilizing cells based on freeze-fracture technology was used to study the distribution of microtubules in wild-type and mutant microspores. Here we show that an abnormal distribution of micro-tubules is correlated with changes in chromosome mor-phology in a cell cycle-dependent manner in po, ms4 and ms6 mutant alleles. After meiosis II, the cell cycle is com-plete and becomes progressively less synchronous in po homozygotes compared with wild-type cells. During microsporogenesis, the distribution of microtubules is abnormal, and chromosome morphology is altered in both po, ms4 and ms6 mutants. However, more chromosome fragments or micronuclei associated with mini-spindles are present in ms6 than po and ms4. After microspores are released from the tetrads, disruptions in structure and organization of chromosomes and microtubules continues in subsequent abnormal cell cycles. However, these cell cycles are incomplete since phragmoplasts are not formed. During these incomplete cell cycles, abnormal spindles and microtubule arrays are induced and extra microtubule arrays are associated with irregularly distributed chromosome fragments. States corresponding to interphase, prophase, metaphase and anaphase can be recognized in the mutant microspores. Abnormal cell cycles also occur after female meiosis during ms4 macrospore development. Since only the cell that normally undergoes embryo sac development (the chazal-most cell) under-goes supernumerary divisions this suggests that the po phenotype can be characterized as premature haploid divisions rather than repetition of meiosis II.
A novel method for measuring protein expression at the cell surface
Authors: Stoorvogel Willem , Oorschot Viola , Neve Bernadette 
Published: April 2021

ABSTRACT
All methods described in the literature that allow quantitative measurements of protein expression at the cell surface are applicable to subsets of surface-exposed proteins only. We developed a new method, involving 3,3-diaminobenzidine (DAB) cytochemistry, which allowed determination of cell-surface expression of all plasma membrane proteins measured, in at least three different cell lines. Adherent cells were first brought into sus-pension by proteinase K and EDTA treatment at 0°C removing many, but not all, surface-exposed proteins. Subsequently, horseradish peroxidase (HRP) was linked by means of its glycosyl residues to specific cell-surface-exposed sugar moieties using the multivalent lectin con-canavalin A (ConA). The suspended cells were encap-sulated by polymerized DAB, a process that was catalysed by plasma membrane-bound HRP. After cell lysis, and removal of nuclei and most of the DAB polymer by centrifugation, proteins were analysed by SDS-PAGE. Surface proteins encapsulated by non-pelleted DAB polymer were retained on top of the stacking gel. After 125I-labelling the cell surface, protease-resistant125I-labelled proteins could be quantitatively coupled to DAB polymer. This process was completely dependent on the presence of ConA, HRP, DAB and H2O2. Sur-face 125I-labelled-Na+,K+-ATPase was resistant to proteinase K but could be completely removed using DAB cytochemistry. Intracellular ConA binding proteins were not affected. Other intracellular proteins, includ-ing endosomal asialoglycoprotein receptor and cation-independent mannose 6-phosphate/insulin-like growth factor II receptor were also not affected. Metabolically [35S]methionine-labelled ‘high-mannose’ glycosylated-Na+,K+-ATPase was not touched by DAB cytochemistry whereas complex-glycosylated surface-exposed 35S--Na+,K+-ATPase was removed by the procedure. The results show that the method can be used to measure both endocytic uptake and biosynthetic arrival at the plasma membrane of membrane-associated proteins.
Stacking of Golgi cisternae in Schizosaccharomyces pombe requires intact microtubules
Authors: Ayscough Kathryn , Hajibagheri Nasser M. A. , Watson Rose , Warren Graham 
Published: April 2021

ABSTRACT
Fission yeast was treated with the anti-microtubule agent, thiabendazole. Cytoplasmic microtubules broke down with a half-time of less than 10 minutes followed closely by the unstacking of Golgi cisternae. The final product appeared to be single Golgi cisternae. No other organelle seemed to be affected by this treatment, which was completely reversible. The nda3 mutant strain has an alteredtubulin and its cytoplasmic microtubules are resistant to thiabendazole. The Golgi in this cold-sensitive mutant was unaffected by treatment at the per-missive temperature but unstacked at the non-permissive temperature even in the absence of thiabendazole. Taken together these data show that disruption of the microtubular network can cause dissociation of Golgi cisternae. Newly synthesised acid phosphatase was transported and secreted to the same extent and with the same kinetics whether or not the Golgi was unstacked. The possible role of microtubules in Golgi stacking and the lack of effect on secretion are discussed.
Transient expression of the intermediate filament nestin during skeletal muscle development
Authors: Sejersen Thomas , Lendahl Urban 
Published: April 2021

ABSTRACT
It has previously been established that skeletal muscle development is accompanied by changes in the compo-sition of intermediate filaments: vimentin is expressed predominantly in myoblasts and desmin in adult myotubes. We show that the intermediate filament tran-sitions during muscle development are more complex, and involve a transient expression of the recently dis-covered intermediate filament nestin. Nestin RNA is expressed predominantly early, in a biphasic pattern, and is markedly downregulated in adult rat muscle, whereas desmin RNA becomes more abundant through-out development. Nestin protein was found up to the postnatal myotube stage, where it colocalized with desmin in Z bands. The intracellular distribution of nestin, vimentin and desmin was analysed in the human myogenic cell line G6 before and after in vitro differ-entiation. Despite its more distant evolutionary and structural relationship to the other two intermediate fil-aments, nestin formed a cytoplasmic filamentous net-work indistinguishable from that of desmin and vimentin, both in undifferentiated myoblasts and after differentiation to multinuclear myotubes. In conclusion, our data suggest that nestin is an integrated component of the dynamic intermediate filament network during muscle development and that nestin copolymerizes with desmin and vimentin at stages of coexpression.
Contribution of melanogenic proteins to the heterogeneous pigmentation of human melanocytes
Authors: Abdel-Malek Zalfa , Swope Viki , Collins Cynthia , Boissy Raymond , Zhao Huiquan , Nordlund James 
Published: April 2021

ABSTRACT
Human melanocytes from individuals with different skin types, as well as from the skin of the same indi-vidual, are heterogeneous in their melanin content. This heterogeneity may be attributed to differences in the activity and expression of the three melanogenic proteins: tyrosinase and tyrosinase-related proteins 1 and 2 (gp75 and DOPAchrome tautomerase, respectively), which in turn are affected by certain regulatory factors. Established melanocyte strains that exhibited intrinsic melanogenic heterogeneity could be separated into sub-populations according to density and melanin content by Percoll density gradient centrifugation. The least melanotic subpopulation consisted of melanocytes that contained an active tyrosinase enzyme and a low amount of melanin. Tyrosinase activity and the quantities of tyrosinase enzyme, tyrosinase-related protein-1 and DOPAchrome tautomerase gradually increased with increased melanin content and Percoll density of the isolated melanocyte subpopulations. We have found a direct correlation between melanin content, tyrosinase activity and the expression of the three melanogenic pro-teins in melanocyte strains established from different skin types. Addition of the two epidermal cytokines, tumor necrosis factor- or interleukin-1, to cultures of human melanocytes from different skin types caused decreased proliferation, tyrosinase activity and expression of tyrosinase, tyrosinase-related protein-1 and DOPAchrome tautomerase. Similar results were obtained when Percoll-derived melanocyte subpopula-tions were treated with tumor necrosis factor- and interleukin-1. These results indicate that the variation in melanin content in human melanocytes is due to differences in the activity and expression of the melanogenic proteins, which are influenced by autocrine and paracrine factors.
Ultrastructural rRNA localization in plant cell nucleoli RNA/RNA in situ hybridization, autoradiography and cytochemistry
Authors: Olmedilla A. , Testillano P. S. , Vicente O. , Delseny M. , Risueño M. C. 
Published: April 2021

ABSTRACT
The distribution of ribosomal transcripts in the plant nucleolus has been studied by non-isotopic in situ hybridization in ultrathin Lowicryl K4M sections and by high-resolution autoradiography after labelling with tritiated uridine. In parallel, cytochemical techniques were applied to localize RNA on different plant nucle-olar components of Allium cepa L. root meristematic cells and Capsicum annuum L. pollen grains.
For RNA/RNA in situ hybridization, several biotiny-lated single-stranded ribosomal RNA probes were used for mapping different fragments of the 18 S and the 25 S rRNA gene transcribed regions. Ribosomal RNAs (from pre-rRNAs to mature 18 and 25 S RNAs) were found in the nucleolus, in the dense fibrillar (DFC) and granular components (GC). Hybridization signal was found at the periphery of some fibrillar centres (FCs) with probes recognizing both 18 and 25 S rRNA sequences. A quantitative study was performed to analyze the significance of this labelling.
Incorporation of tritiated uridine into roots was car-ried out and, later, after a long time-exposure, autora-diography revealed the presence of newly synthesized RNA mainly in the DFC and at the periphery of the FCs. The presence of RNA in these areas was also con-firmed by the cytochemical techniques used in this study.
Taken together, these data favour the hypothesis that transcription can begin at the periphery of the FCs, although we cannot exclude the possibility that the DFC plays a role in this process.
Intracellular distribution of β-actin mrna is polarized in embryonic corneal epithelia
Authors: Yeh Berhan , Svoboda Kathy Kay Hartford 
Published: April 2021

ABSTRACT
The intracellular distribution of filamentous actin (F-actin), all actin isoforms and β-actin mRNA were analyzed in whole-mount preparations of freshly isolated corneal epithelia. Filamentous actin distribution was analyzed with fluorescently tagged phalloidin. An antibody that recognizes an epitope on both globular (G-actin) and F-actin was used in an immunohistochemical analysis of actin protein distribution. Whole-mount epithelial tissues were examined with a confocal laser scanning microscope (CLSM). Biotinylated oligonucleotide probes specific for the β-actin mRNA were used, and visualized with avidinFITC. The intracellular localization of the β-actin mRNA was similar to the F-actin protein distribution. In the most apical optical sections of embryonic cornea, actin staining delineated the cell borders and microvilli of the periderm cells. The actin is also detected as an organized network at the interface between the basal and periderm cells. At the level of the basal cell nucleus, F-actin is sparse, associating only with the lateral cell membranes. However, at the optical plane below the nuclei, the actin forms an elaborate actin cortical mat. Actin mRNA staining was visualized as discrete punctate areas. The β-actin mRNA was positive at the optical plane just below the periderm cell apical membrane surface, similar to actin in microvilli. These cells also contained punctate staining near the cell membranes and in the periderm-basal cell junction area. At the level of the basal cell nucleus the actin mRNA was present in a punctate pattern along the cell membranes. Below the basal cell nuclei the actin mRNA staining increased at the level of the actin cortical mat. These experiments are the first demonstration that actin mRNA is polarized in embryonic corneal epithelia and co-localized with actin protein in an intact tissue.
Evidence for retrograde traffic between terminal lysosomes and the prelysosomal/late endosome compartment
Authors: Jahraus Andrea , Storrie Brian , Griffiths Gareth , Desjardins Michel 
Published: April 2021

ABSTRACT
We have investigated the interactions occuring between the prelysosomal compartment, PLC/late endosome, and terminal lysosomes using an approach that allowed us to internalize and deliver specific tracers to these compartments, and look for evidence of their meeting. After internalization of sucrose, the lysosomes (sucrosomes), but not the PLC/late endosomes, became significantly swollen and visible in the light microscope. If invertase is then added to the medium it reaches the lysosomes where it cleaves sucrose into fructose and glucose. These sugars, unlike sucrose, can be transported into the cytosol, resulting in the disappearance of the sucrosomes. We previously showed that phagocytosed latex beads are delivered specifically to, and reside in, the PLC/late endosome, a stage earlier than the lysosomes in the endocytic pathway (Rabinowitz et al. (1992)J. Cell Biol. 116, 95-112). In the present study, we demonstrate that invertase conjugated to the latex beads, and thus immobilized in late endosomes, has access to the sucrose present in the more distal lysosomes. Experiments using nocodazole indicate that this retrograde fusion event requires the presence of an intact microtubule network. The simplest interpretation of our results is that the two compartments fuse, allowing for a retrograde transport of sucrose from the lysosomes to the PLC/late endosomes.
Merosin is synthesized by thyroid cells in primary culture irrespective of cellular organization
Authors: Andre Frédéric , Filippi Pierre , Feracci Hélène 
Published: April 2021

ABSTRACT
The in vitro synthesis and deposition of laminin family gly-coproteins were studied using primary porcine thyroid cells cultured as monolayers or in follicles. The latter organization mimics the in vivo state of these polarized epithelial cells. In both cell systems a trimeric molecule was immunoprecipitated by using polyclonal antibodies against EHS-laminin. When the cells were fully polarized the protein was found at the basal pole of cells, irrespective of their organization. However, this molecule was different from laminin purified from a traditional source, the murine Engelbreth-Holm-Swarm (EHS) tumor. Thyroid cell laminin was composed of two light chains, analogous to EHS B1 and B2, and a disulfide-bonded heavy chain not found in EHS-laminin. The heavy chain was first synthe-sized as a 380 kDa polypeptide, then rapidly cleaved to a doublet of 350-380 kDa, which was subsequently found in both cell extracts and conditioned culture media. This thyroid laminin variant was compared with merosin, another variant found in the basement membranes of tro-phoblast, Schwann cells, striated muscle and liver. The heavy chain (M) of merosin shows homology to EHS-laminin heavy chain at the C-terminal domain, and is usually found as two polypeptides of 80 kDa and 300 kDa (Ehrig K., Leivo I., Argraves W. S., Ruoslahti E. and Engvall E. Proc. Nat. Acad. Sci. 87, 3264-3268, 1990). mRNA of the M chain was identified by RT-PCR in freshly isolated thyrocytes as well as in 6-day-old cultured thyroid cells. Furthermore, both the classical laminin heavy chain and the 350 kDa variant were detected by immunoblotting and immunofluorescence in the thyroid gland in vivo. All these results suggest strongly that merosin is a basement membrane component of thyroid cells in vivo and in vitro.
An amino-terminal domain of the growth-associated protein gap-43 mediates its effects on filopodial formation and cell spreading
Authors: Strittmatter Stephen M. , Valenzuela Dario , Fishman Mark C. 
Published: April 2021

ABSTRACT
GAP-43 is a neuronal protein that is believed to be important to neuronal growth and nerve terminal plastic-ity. It is enriched on the inner surface of growth cone membranes, a localization that may depend upon palmi-toylation of Cys3 and Cys4. It is a major substrate for protein kinase C, which phosphorylates Ser41. Isolated GAP-43 can bind to actin and to calmodulin, and can activate the heterotrimeric GTP-binding proteins, Go and Gi. A peptide consisting of the GAP-43 sequence 39-55 binds calmodulin, and an amino-terminal GAP-43 (1-10) peptide activates Go, suggesting that these stretches may be functional domains of the intact protein.
When expressed in non-neuronal cells, GAP-43 enhances filopodial extension and has effects upon cell spreading. We have examined the effects of various GAP-43 domains upon this assay, by expression of GAP-43, GAP-43 mutant proteins, and GAP-43-CAT fusion proteins in COS-7 cells. We find that the amino terminus (Met-Leu-Cys-Cys-Met-Arg-Arg-Thr-Lys-Gln) is an important contributor to these effects on cell shape. A GAP-43 protein mutant in Cys3 and Cys4 does not bind to the membrane, and is inactive. Mutants in Arg6 or Lys9 also are inactive, although they remain localized to particulate fractions; Arg7 mutants are active. A chimeric gene consisting of GAP-43 (1-10) fused to chloramphenicol acetyl transferase (CAT) also causes cell shape changes. As for GAP-43, the effects of this fusion protein are abolished by mutations of Cys3, Cys4, Arg6 or Lys9, but not by mutation of Arg7. Therefore, the cell surface activity of transfected GAP-43 depends upon its amino terminus, although other domains may regulate it in this regard.
Since the amino-terminal domain includes the peptide stretch known to be capable of activating Go and Gi, we examined the effect of GAP-43 on a Gi-regulated second messenger system, the inhibition of cAMP production in A431 cells. A431 cells stably transfected with GAP-43 spread less well than do controls. In addition, they evidence decreased levels of forskolin-stimulated cAMP, consistent with chronic stimulation of Gi. Stimulation of adenylate cyclase by isoproterenol reverses the GAP-43-induced changes in cell shape. This suggests that G protein stimu-lation is involved in GAP-43 effects upon cell shape.