bluuebunny/crossref_metadata_2025_split huggingface dataset:
Demonstration of cell cycle kinetics in thyroid primary culture by immunostaining of proliferating cell nuclear antigen: differences in cyclic AMP-dependent and -independent mitogenic stimulations
Authors: Baptist Mireille , Dumont Jacques E. , Roger Pierre P. 
Published: April 2021

ABSTRACT
In this study, experimental conditions are described that allowed us to follow the fate of the DNA polymerase 3-associated proliferating cell nuclear antigen (PCNA), by immunolabeling during the overall cell cycle. Differences in subcellular localization or the presence of PCNA allowed us to identify each phase of the cell cycle. Using these cell cycle markers in dog thyroid epithelial cells in primary culture, we found unexpected differences in cell cycle kinetics, in response to stimulations through cAMP-dependent and cAMP-independent pathways. These provide a new dimension to the view that the two pathways are largely separate, but co-operate on DNA synthesis initiation. More precisely, thyrotropin (TSH), acting via cAMP, exerts a potent triggering effect on DNA synthesis, associated with a precocious induction of PCNA appearance. This constitutes the major influence of TSH (cAMP) in determining cell cycle progression, which is only partly moderated by TSH-dependent lengthening of S- and G2-phases.
immunolocalisation of collagenase in rabbit periosteal tissue explants and extraction of the enzyme: the effect of the cytokines IL-1α and EGF
Authors: van der Zee Erwin , Everts Vincent , Hoeben Kees , Beertsen Wouter 
Published: April 2021

ABSTRACT
The effect of interleukin-1α (IL-1α) and murine epidermal growth factor (EGF) on incorporation of endogenously produced collagenase in the extracellular matrix of soft connective tissue was studied in an in vitro model system using periosteal explants obtained from rabbit calvariae. Immunohistochemical analysis indicated the highest level of collagenase in explants cultured for 72 hours with IL-1α in combination with EGF. Most enzyme appeared to be associated with the extracellular matrix, but labeling was also found in numerous fibroblast-like cells. Explants cultured in the presence of IL-1α alone contained less enzyme and in periostea treated without cytokines, or with EGF alone, only a faint label, if any, was seen. Freshly isolated, non-cultured periostea contained no detectable enzyme.
Extraction of collagenase from periostea revealed that: (1) non-cultured periosteum did not contain detectable levels of enzyme. (2) The amount of total activatable enzyme synergistically increased (10-fold) under the influence of IL-1α and EGF, whereas IL-1α alone showed a 4-fold enhancement compared to control or EGF-incubated explants. (3) The latent fraction of the enzyme was synergistically increased (up to 100-fold or more) in periostea cultured in the presence of IL-1α+EGF (21.17 mU/explant versus 0.05 mU/explant in controls). (4) Active collagenase, on the other hand, appeared to be present in a relatively high concentration in explants cultured without cytokines (2.45 mU/explant versus 0.36 mU/explant in IL-1α+EGF-treated explants). (5) No enzyme could be extracted from devitalized explants that were incubated in media containing high levels of latent collagenase. It was concluded that collagenase had been incorporated somehow in the extracellular matrix during culturing. In an attempt to analyze breakdown of collagen the amount of hydroxyproline in culture media was assessed. The level of this imino acid released by periostea treated without cytokines or with EGF alone was significantly higher than by those incubated with IL-1α and EGF.
In conclusion, incorporation of collagenase in periosteal connective tissue seems to occur during culturing; the level of the incorporated latent enzyme being strongly enhanced by a combination of the cytokines IL-1α and EGF. However, as the amount of collagen breakdown had decreased, these findings indicate that IL-1α in combination with EGF is able to induce deposition of a large reservoir of latent collagenase in the extracellular matrix, but does not stimulate adequate activation mechanisms to result in enhanced degradation.
A 28 kDa sarcolemmal antigen in kidney principal cell basolateral membranes: relationship to orthogonal arrays and MIP26
Authors: Verbavatz Jean-Marc , Van Hoek Alfred N. , Ma Tonghui , Sabolic Ivan , Valenti Giovanna , Ellisman Mark H. , Ausiello Dennis A. , Verkman A. S. , Brown Dennis 
Published: April 2021

ABSTRACT
Two recently cloned water channels, CHIP28 and WCH-CD, are homologous to MIP26, an integral membrane channel-forming protein found in lens fiber plasma membranes. CHIP28 is found in basolateral and apical plasma membranes of kidney proximal tubules and thin descending limbs of Henle, whereas WCH-CD is apically located in collecting duct principal cells. So far, the putative water channel that may be responsible for the high constitutive permeability of principal cell basolateral membranes has not been identified. Interestingly, freeze-fracture electron microscopy has shown that characteristic orthogonal arrays of intramembrane particles (OAPs) are found on the basolateral plasma membranes of collecting duct principal cells, and that morphologically identical OAPs present in lens fiber cell plasma membranes contain the protein MIP26. Similar OAPs have also been detected on plasma membranes of other cell types including gastric parietal cells, astroglial cells and skeletal muscle fibers. By indirect immunofluorescence, western blotting and northern blotting, MIP26 was found only in lens fibers. In addition, functional studies on reconstituted and oocyte-expressed MIP26 excluded the possibility that MIP26 might be a basolateral water channel in the kidney. However, a polyclonal antibody raised against skeletal muscle sarcolemmal vesicles, which are enriched in OAPs, produced an intense staining of principal cell basolateral plasma membranes in kidney collecting duct and immunoprecipitated a 28 kDa protein from kidney papilla. The immunoprecipitated protein from papilla was not recognized by anti-CHIP28 or anti-MIP26 antibodies, indicating that principal cell basolateral membranes contain a novel member of the CHIP/MIP family. Because this antibody also stained brain astrocyte end feet, which are enriched in OAPs, it is possible that the 28 kDa protein is related to these structures. We conclude that OAPs probably contain related but distinct proteins that may have different membrane channel functions in different cell types.
Cytoskeleton-dependent transport of cytoplasmic particles in previtellogenic to mid-vitellogenic ovarian follicles of Drosophila: time-lapse analysis using video-enhanced contrast microscopy
Authors: Bohrmann Johannes , Biber Knut 
Published: April 2021

ABSTRACT
In Drosophila oogenesis, several morphogenetic determinants and other developmental factors synthesized in the nurse cells have been shown to accumulate in the oocyte during pre- to mid-vitellogenic stages. However, the mechanisms of the involved intercellular transport processes that seem to be rather selective have not been revealed so far. We have investigated in vitro, by means of video-enhanced contrast time-lapse microscopy, the transport of cytoplasmic particles from the nurse cells through ring canals into the oocyte during oogenesis stages 6-10A. At stage 7, we first observed single particles moving into the previtellogenic oocyte. The particle transfer was strictly unidirectional and seemed to be selective, since only some individual particles moved whereas other particles lying in the vicinity of the ring canals were not transported. The observed transport processes were inhibitable with 2,4-dinitrophenol, cytochalasin B or N-ethylmaleimide, but not with microtubule inhibitors. At the beginning of vitellogenesis (stage 8), the selective translocation of particles through the ring canals became faster (up to 130 nm/second) and more frequent (about 1 particle/minute), whereas during mid-vitellogenesis (stages 9-10A) the velocity and the frequency of particle transport decreased again. Following their more or less rectilinear passage through the ring canals, the particles joined a circular stream of cytoplasmic particles in the oocyte. This ooplasmic particle streaming started at stage 6/7 with velocities of about 80 nm/second and some reversals of direction at the beginning. The particle stream in the oocyte was sensitive to colchicine and vinblastine, but not to cytochalasin B, and we presume that it reflects the rearrangement of ooplasmic microtubules described recently by other authors. We propose that during stages 7-10A, a selective transport of particles into the oocyte occurs through the ring canal along a polarized scaffold of cytoskeletal elements in which microfilaments are involved. This transport might be driven by a myosin-like motor molecule. Either attached to, or organized into, such larger particles or organelles, specific mRNAs and proteins might become selectively transported into the oocyte.
Sense and antisense transfection analysis of tau function: tau influences net microtubule assembly, neurite outgrowth and neuritic stability
Authors: Esmaeli-Azad Babak , McCarty Joseph H. , Feinstein Stuart C. 
Published: April 2021

ABSTRACT
Microtubules are fundamental elements participating in many aspects of cell behavior and maintenance, yet the factors regulating microtubule behavior in vivo remain poorly understood. Employing the nerve growth factor (NGF)-responsive cell line, PC12, we have used sense and antisense DNA transfection strategies to examine the role of the microtubule-associated protein (MAP) tau in several aspects of neuronal cell behavior. Stable transfectants over-expressing tau accumulate more microtubule mass and extend neurites more rapidly than control cells, while transfectants under-expressing tau exhibit reduced micro-tubule levels and slower neurite outgrowth. Further, tau over-expressing cells are markedly more resistant to nocodazole-induced neuritic degeneration when compared to wild-type or tau under-expressing cells. These observations provide direct support for the model that tau is capable of influencing: (i) net microtubule assembly, (ii) the rate of neurite elongation and (iii) neuritic stability. These capabilities suggest that tau plays crucial roles in the development and maintenance of neuronal cells.
Quantitative determination of the proportion of microtubule polymer present during the mitosis-interphase transition
Authors: Zhai Ye , Borisy Gary G. 
Published: April 2021

ABSTRACT
We have developed a new method for determining levels of tubulin polymer, based on quantitative fluorescence detection of x-rhodamine tubulin microinjected into living cells and we have applied this method to analysis of the mitosis-interphase transition. LLC-PK cells in interphase and mitosis were microinjected, then cooled and rewarmed to drive tubulin incorporation. Total tubulin fluorescence in individual, living cells was quantified using a cooled, scientific grade CCD image sensor. Cells were then washed and lysed into a microtubule-stabilizing buffer to extract the soluble pool. Total tubulin polymer fluorescence was determined for the extracted cells in the same way as for living cells. Fluorescence images were corrected by flatfielding and background subtraction. The ratio of extracted cell fluorescence/living cell fluorescence for individual cells, was taken as the proportion of tubulin as polymer. Cells in M-phase, G1 and random interphase were analyzed. G1 cells had almost the same proportion as random interphase cells. Mitotic cells gave a value of 90±5% of G1 cells at 37°C. Within M-phase, levels of tubulin as polymer in metaphase and early anaphase were not significantly different. In contrast to the general expectation of micro-tubule depolymerization at anaphase onset, these results indicate that as cells exit mitosis, the overall proportion of tubulin as polymer does not change dramatically even though the mitotic spindle disassembles. We conclude that the mitosis-interphase transition is accompanied by a redistribution of tubulin at an essentially constant polymer level. Therefore, a global shift to depolymerization conditions is not the driving force for anaphase chromosome movement.
Development of a spontaneous permanent cell line of rabbit corneal epithelial cells that undergoes sequential stages of differentiation in cell culture
Authors: Castro-Muñozledo Federico 
Published: April 2021

ABSTRACT
Established epithelial cell lines that retain their differen-tiation potential and growth regulatory characteristics can provide valuable tools for studying gene regulation, extra-cellular matrix synthesis or growth factor response. They are also useful for drug development and toxicity testing. Experiments were therefore carried out to optimize culture conditions for the long-term, serial transfer of corneal epithelial cells in the presence of 3T3 feeder layers; and to establish a permanent cell line. In such experiments, rabbit corneal epithelial cells were seeded at low inoculation densities, and transferred every 5 days. After 80 popula-tion doublings, an epithelial cell line, RCE1, emerged. The cell line is heteroploid, with an average population doubling time of 15.5 hours (vs 18 hours for primary cultures). When RCE1 cells reached confluence, they strat-ified to form a three-to five-layered epithelium and expressed the differentiation-related keratin pair K3/K12 as shown by immunoblot and immunostaining. Biosyn-thetic labeling of proliferating, confluent and stratified cultures further showed that RCE1 cells expressed keratin pairs K5/K14, K6/K16 and K3/K12, thus mimicking faith-fully the stage-dependent differentiation of primary cultures of rabbit corneal keratinocytes. The results demonstrated that RCE1 cells provide a useful model for studying corneal cell growth and differentiation.
Ca2+ Signaling in the Chlamydomonas flagellar regeneration system: cellular and molecular responses
Authors: Cheshire J. L. , Evans J. H. , Keller L. R. 
Published: April 2021

ABSTRACT
In response to certain extracellular stimuli, Chlamydomonas reinhardtii cells excise their flagella, induce expression of more than 200 different flagellar mRNAs, and assemble a new flagellar pair. Normally, flagellar excision, gene induction and outgrowth are tightly coupled temporally. Our previous studies showed that uncoupling the cellular response of flagellar excision from flagellar outgrowth resulted in submaximal flagellar gene induction, and led us to propose that normal flagellar gene induction is a composite response. The present study extends these observations by measuring flagellar gene induction in Chlamydomonas cells stimulated under conditions where both flagellar excision and flagellar outgrowth are blocked. We find that the flagellar genes are induced in a Ca2+-dependent manner in response to stimulation in the absence of flagellar excision and outgrowth. Flagellar gene induction is therefore independent of flagellar excision and outgrowth but sensitive to extracellular Ca2+ levels. Thus, flagellar excision, flagellar outgrowth and flagellar gene induction are three responses to a common stimulus that are related by their requirement for extracellular Ca2+
Astrocytes modulate retinal vasculogenesis: effects on fibronectin expression
Authors: Jiang B. , Liou G. I. , Behzadian M. A. , Caldwell R. B. 
Published: April 2021

ABSTRACT
Vasculogenesis is the formation of blood-vessels by differentiation of vascular precursor cells. Experiments using retinal models were designed to test the hypothesis that astrocytes influence this process by effects on the composition of the extracellular matrix. Retinal vasculogenesis was studied in relation to the migration of astrocytes and expression of the extracellular matrix proteins laminin and fibronectin by in vivo experiments in neonatal rats. The results show that astrocytes spread into the retina just ahead of the newly formed vessels, where they probably initiate vasculogenesis. They also establish that fibronectin, but not laminin, is expressed in the zone of vasculogenesis immediately prior to vessel formation. Increased amounts of fibronectin mRNA indicate that fibronectin is synthesized by cells within this same region during this same time period. Later, as the new vessels form, differentiation of endothelial cells is correlated with the appearance of pericytes in the vessel wall and laminin in the vascular basement membrane. In vitro experiments using conditioned medium approaches showed that astrocytes stimulate endothelial cell fibronectin expression. Taken together with the in vivo observations these in vitro results suggest that fibronectin expression is an essential component in the initiation of retinal vasculogenesis. This study is the first indication that astrocytes influence the fibronectin component of the extracellular matrix during retinal vasculogenesis and that expression of fibronectin precedes that of laminin in this process.
The effects of diazepam on mitosis and the microtubule cytoskeleton I. Observations on the diatoms Hantzschia amphioxys and Surirella robusta
Authors: Spurck Timothy P. , Pickett-Heaps Jeremy D. 
Published: April 2021

ABSTRACT
The effects of diazepam (DZP) on mitosis and the micro-tubule (MT) cytoskeleton in the live diatoms Hantzschia amphioxys and Surirella robusta were followed using time-lapse video microscopy. Similarly treated cells were fixed and later examined for immunoflouresence staining of MTs or for transmission electron microscopy.
DZP treatment (250 μM) had no effect on interphase cells but affected mitosis, resulting in the majority of prometaphase and metaphase chromosomes releasing from one or both spindle poles and collecting irregularly along the central spindle. Chromosomes remaining attached to one pole continued to display slight prometaphase oscillations; however, this activity was never observed in metaphase spindles. Following removal of DZP, some chromosomes still bipolarly attached, immediately released elastically from one pole. Within the first 2 minutes of recovery, all chromosomes recommenced spindle attachment, exhibiting normal prometaphase oscillations and proceeded through mitosis. DZP treatment during anaphase had no detectable effect on chromosome motion or cell cleavage.
These results suggest that DZP acts as an anti-MT agent, selectively affecting polar MTs at prophase, prometaphase and metaphase, and thereby weakening kinetochore connection to the poles. From these and other results (unpub-lished), its mode of action is different to that of most anti-MT agents.
Cell-to-cell adherens junction formation and actin filament organization: similarities and differences between non-polarized fibroblasts and polarized epithelial cells
Authors: Yonemura Shigenobu , Itoh Masahiko , Nagafuchi Akira , Tsukita Shoichiro 
Published: April 2021

ABSTRACT
Cadherin has an intimate spatial relationship with actin filaments (AF) in various types of cells, forming the cell-to-cell adherens junction (AJ). We compared the AJ/AF rela-tionship between non-polarized fibroblasts (NRK cells) and polarized epithelial cells (MTD-1A cells). E/P-cadherin, α-catenin, ZO-1 and vinculin were localized with reference to AF in these cells using laser scan microscopy as well as conventional light and electron microscopy. NRK cells adhered to each other at the tips of thin cellular processes, where spot-like AJ were formed, where P-cadherin, α-catenin, ZO-1 and vinculin were concentrated. Some stress-fiber-like AF bundles ran axially in these processes and terminated at spot-like AJ on their tips. At the electron micro-scopic level these spot-like AJ were seen as aggregates of small ‘units’ of AJ, where AF were densely and perpendicularly associated with the plasma membrane. In MTD-1A cells, the AJ/AF relationship was investigated during the cell polarization process after replating or wounding. At the early stage, the AJ/AF relationship was quite similar to that in NRK cells. As polarization proceeded, the spot-like AJs were gradually fused side by side with the concomitant shortening of the associated stress-fiber-like AF bundles. Finally, the belt-like AJ was established, which was lined with circumferential AF bundles. The similarities and differences in the AJ/AF relationship between non-polarized fibroblasts and polarized epithelial cells are discussed.
The exit of mouse oocytes from meiotic M-phase requires an intact spindle during intracellular calcium release
Authors: Winston Nicola J. , McGuinness Orla , Johnson Martin H. , Maro Bernard 
Published: April 2021

ABSTRACT
To study the role of the metaphase spindle during the period of oocyte activation, mouse oocytes were fertilised or activated parthenogenetically in the presence or absence of the microtubule inhibitor nocodazole. In both cases, nocodazole caused the disappearance of the spindle and prevented the passage of the oocytes into interphase. However, the calcium spiking responses of the oocytes were not affected by nocodazole, being repetitive after fertilisation and a single spike after activation. If, after their activation or fertilisation in nocodazole, oocytes were later removed from the drug, only those that had been fertilised progressed into interphase. This progress was associated with continuing calcium spiking. Moreover, both the spiking and the progress to interphase could be blocked or reduced in incidence by removal of external calcium or addition of 5,5′-dimethyl BAPTA-AM. Oocytes that had been activated by ethanol in the presence of nocodazole and then removed from it, to allow reformation of the spindle, only progressed into interphase if given a second exposure to ethanol, thereby eliciting a second calcium transient. These results show that exit from meiotic M-phase requires the simultaneous presence of a fully intact spindle during the release of calcium and that those factors leading to the degradation of cyclin B are only activated transiently. Since cyclin is being degraded continuously in the metaphase-II-arrested mouse oocyte and since this degradation is micro-tubule-dependent, these data suggest that the superimposition of a high concentration of intracellular calcium is required to tilt the equilibrium further in favour of cyclin degradation if exit from M-phase is to occur.