bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mitotic block in HeLa cells by vinblastine: ultrastructural changes in kinetochore-microtubule attachment and in centrosomes
Authors: Wendell Kim Livezey , Wilson Leslie , Jordan Mary Ann 
Published: April 2021

ABSTRACT
Previous work from this laboratory has indicated that very low concentrations of vinblastine block HeLa cells at mitosis in the presence of a full complement of microtubules and without major disruption of spindle organization. In the present study we analyzed the structural organization of mitotic spindle microtubules, chromosomes and centrosomes by electron microscopy after incubating HeLa cells for one cell cycle with 2 nM vinblastine. We found that mitotic block of HeLa cells by vinblastine was associated with alterations of the fine structure of the spindle that were subtle but profound in their apparent consequences. The cell cycle was blocked in a stage that resembled prometaphase or metaphase; chromosomes had not undergone anaphase segregation. Neither the structure of the microtubules nor the structure of the kinetochores was detectably altered by the drug. However, the number of microtubules attached to kinetochores was decreased significantly. In addition, the centrosomes were altered; the normal close association of mother and daughter centriole was lost, numerous membranous vesicles were found in the centrosomal region, and many centrioles exhibited abnormal ultrastructure and had microtubules coursing through their interiors. These findings are consistent with our previous results and indicate that inhibition of the polymerization dynamics of mitotic spindle microtubules and perhaps of centriole microtubules, rather than microtubule depolymerization, is responsible for the mitotic inhibition by vinblastine.
Induction of tenascin in cancer cells by interactions with embryonic mesenchyme mediated by a diffusible factor
Authors: Hiraiwa Noriko , Kida Hirokatsu , Sakakura Teruyo , Kusakabe Moriaki 
Published: April 2021

ABSTRACT
Human cancer cell lines A431 and MCF7, which do not produce tenascin (TN) in vitro, were found to produce TN when injected into nude mice or co-cultured with the embryonic mesenchyme. The TN expression in the developing A431 solid tumor was demonstrated by immunohistochemistory and by in situ hybridization. Human TN was detected in culture media by western blot analysis using human specific monoclonal antibody (RCB-1). During tumorigenesis, in the early stage, mouse TN was actively induced and deposited in the peri- and intertumor spaces surrounding the developing tumor. Two days later, TN derived from human epithe- lial cancer cells was induced and mainly deposited in the intertumor basement membrane. After this stage, tumor cells were actively producing TN. On the other hand, TN induction in non TN-producing cells, such as A431 and MCF7 cell lines, was also observed in vitro. Although cell lines such as NIH-3T3, φ2 , STO, 2H6, 3E5 and CMT315, had no effect on the TN induction, primary cultured embryonic mesenchyme effectively stimulated the TN expression in the cancer cell lines. This mesenchymal effect decreased with age and was entirely lost postnatally. Furthermore, conditioned media from these embryonic mesenchymes could repro- duce the same effects on TN induction as observed in the co-culture study.
In conclusion, these findings suggest that TN induc- tion in epithelial cancer cells may depend on interac- tions with the surrounding environment, that these interactions may be mediated by a soluble factor(s) derived from the surrounding mesenchyme and that the TN induction observed in the tumorigenesis may reflect histogenesis during the embryonic period.
Non-genotoxic hepatocarcinogenesis in vitro: the FaO hepatoma line responds to peroxisome proliferators and retains the ability to undergo apoptosis
Authors: Bayly Alison C. , French Nicola J. , Dive Caroline , Roberts Ruth A. 
Published: April 2021

ABSTRACT
A range of hepatoma cell lines (RH1, HTC, FaO, 7800C1 and MH1C1), has been studied with the aim of estab- lishing an in vitro model to investigate the molecular mechanisms of hepatocarcinogenicity induced by the peroxisome proliferator class of non-genotoxic carcino- gens. In view of speculation that peroxisome prolifera- tors suppress hepatocyte apoptosis in vivo, we have placed particular emphasis on evaluating whether hepatoma cell lines retain the ability to undergo apop- totic cell death.
Expression of the liver-specific differentiation marker albumin and the peroxisome proliferator-activated receptor (PPAR) was highest in the Reuber hepatoma cell line, FaO. This cell line also demonstrated the most marked response to the peroxisome proliferator nafenopin with a 2.2-fold induction of the microsomal enzyme cytochrome p450IVA1. This response was found to display intercellular heterogeneity by immunocyto- chemistry. Thus, the FaO cell line maintained charac- teristics of hepatocytes, both in vivo and in vitro, in terms of expression of constitutive and inducible mark- ers. However, none of the cell lines tested mirrored the hyperplastic response of hepatocytes to nafenopin, since no increase in cell growth kinetics was observed on addition of nafenopin to the growth medium.
The mode of cell death in confluent FaO cultures was characterised as apoptosis, by fluorescence microscopy and agarose gel electrophoresis of extracted DNA. Cells detaching from confluent FaO cultures exhibited chro- matin condensation and DNA fragmentation patterns characteristic of cells undergoing apoptotic death. Inter- estingly, no apoptosis was seen in monolayer cells, sug- gesting that apoptosis in vitro is associated with cell shrinkage and detachment similar to that documented for the liver in vivo. The FaO cell line may be a useful tool to investigate the hypothesis that the suppression of apoptosis plays a crucial role in peroxisome prolifera- tor-induced hepatocarcinogenesis.
Quantitative analysis of stress fiber orientation during corneal wound contraction
Authors: Petroll W. Matthew , Cavanagh H. Dwight , Barry Patricia , Andrews Peter , Jester James V. 
Published: April 2021

ABSTRACT
Previous studies of actin and actin-binding proteins in corneal myofibroblasts suggest the development of a contractile apparatus composed, in part, of F-actin micro-filament bundles, i.e. stress fibers. To better understand the mechanics of wound contraction and the relationship between microfilament bundles and wound closure, we have analyzed the spatial and temporal organization of stress fibers during the process of corneal wound healing. Rabbit corneas (26 eyes) received 6 mm full-thickness, central incisions and were studied at various times for F-actin organization using en bloc (whole cornea) staining with FITC-phalloidin, as well as conventional histological techniques. 3-D datasets (z-series of 40 en face optical sections, 1 m steps) were collected using the Biorad MRC-600 laser scanning confocal microscope at various regions within the wound. At 7 days, 3-D analysis showed randomly oriented, interconnected F-actin filament bundles (stress fibers). Between 7 and 28 days, stress fibers appeared to organize gradually into planes parallel to the wound surface, with a large population achieving a final orientation nearly parallel to the long axis of the wound. Using Fourier Transform analysis techniques, an orientation index (OI) was calculated to quantitate global fiber orientation at each time point. Analysis of variance demonstrated a significant change (P ≤ 0.001) in overall stress fiber orientation from a random distribution at day 7 to an alignment more parallel to the lateral wound borders at day 28. Overall, these data suggest that stress fibers undergo temporal changes in spatial organization that correlate with wound closure, and that wound closure does not involve the development of previously described contractile or tractional forces aligned directly across the wound.
CD44 expression on murine tissues
Authors: Kennel Stephen J. , Lankford Trish K. , Foote Linda J. , Shinpock Sarah G. , Stringer Claude 
Published: April 2021

ABSTRACT
CD44 is a cell surface glycoprotein found on lymphoid and epithelial cells. Its primary function on lymphocytes and macrophages is to mediate interaction with endothelium, while its function on epithelial cells is not known. The protein has many different forms, generated by alternative mRNA splicing and by post-translational modification, which may mediate different functions. During previous work on murine lung tumor cells, mAb 133-13A was isolated and shown to recognize a surface glycoprotein, P100, of 90-100 103 Mr. Amino acid sequence analysis of purified P100 indicates that it is CD44. Since few data exist to indicate which forms of CD44 are present in different normal tissues, mAb 13313A was used to analyze CD44 expression in mouse tissue. Quantitative data on the distribution of CD44(P100) in mice show that spleen, thymus, liver, intestine, uterus and choroid of the eye are major sites of expression. In addition, epithelia of adrenals, esophagus and trachea are CD44(P100) positive. Previous work on human cell lines has implicated a high molecular mass (130–160 103 Mr) form of the glycoprotein as the form expressed in epithelial cells and carcinomas. Isolation of CD44 proteins from lymphoid tissues in the mouse indicate that, as in human lymphoid tissue, the low molecular mass form (80–90 103 Mr) is predominately expressed. These data show that both small (∼81 103 Mr) and large forms of the glycoprotein are expressed in basal epithelia of esophagus and trachea and in salivary gland, while only the small form is expressed in epithelium of the adrenal cortex and in the murine lung and mammary carcinomas studied. While these data cannot distinguish between specific splice variants, they show that the large forms of CD44 are minor components in normal tissue and seem to be found only in basal epithelium. The CD44 of low Mr found in epithelial tissues is probably associated with lymphoid cell types in the tissues.
Epithelial cells retain junctions during mitosis
Authors: Baker Janice , Garrod David 
Published: April 2021

ABSTRACT
It has long been known that cells show reduced cell-sub-stratum adhesion during mitosis in tissue culture, but it is not generally known whether cell-cell adhesion is also reduced. Epithelial cells, both in culture and in tissues, are linked together by several different types of inter-cellular junctions. Are these junctions disassembled when epithelial cells divide?
Cultured epithelial cells were fluorescently stained for desmosomes, tight junctions and zonulae adherentes, and large numbers of dividing cells examined by light microscopy. The results suggested that all three types of intercellular junctions were retained throughout cell division and no evidence for internalization of junctions was obtained. The persistence of intercellular junctions by cultured cells during division was confirmed by electron microscopy. In order to determine whether intercellular junctions were similarly retained by dividing cells in tissues, human colonic mucosal crypt cells and basal keratinocytes were studied by electron microscopy. Both cell types retained intercellular junctions during division. Dividing basal keratinocytes also possessed hemidesmosomal contact with the basement membrane. It is suggested that retention of cellular junctions during division is important for maintenance of tissue integrity and organization.
Receptor-mediated endocytosis of transferrin at the blood-brain barrier
Authors: Roberts Richard L. , Fine Richard E. , Sandra Alexander 
Published: April 2021

ABSTRACT
Rat brains were perfused with a transferrin-peroxidase conjugate (Tf-HRP) to characterize morphologically the endocytic pathway of transferrin in blood-brain barrier endothelial cells. Electron microscopic evaluation of rat brains perfused with Tf-HRP at 4°C and subsequently warmed to 37°C for brief periods of time (2 minutes) showed sequestration of Tf-HRP in clathrin coated pits and vesicles on the luminal membrane of the brain endothelium. After 5 minutes of warming, diaminoben-zidine (DAB) reaction product was present in vesicular structures 250μ500 nm in diameter and in associated tubules morphologically identified as large or sorting endosomes. Recycling endosomes were also heavily labelled at this time point. Almost no DAB reaction product remained in the cerebral endothelial cells when the warming period was longer than 15 minutes. Other rat brains were perfused with Tf-HRP at 30°C for 15 minutes prior to fixation and DAB cytochemistry. In these studies, brain endothelial cells contained large amounts of DAB reaction product, mostly localized in 50–100 nm vesicles and tubules, often in the Golgi region of the cells. Coated pits and vesicles and large endo-somes were also heavily labelled. Transcytosis of Tf-HRP was not identified in either perfusion protocol. Ultrastructural, indirect immunocytochemical localization of transferrin receptors showed that the transferrin receptor is highly polarized at the blood-brain barrier and is localized only on the apical membrane, in contrast to other polarized epithelial cells, like hepatocytes, in which the receptor is present on the basolateral membrane. The evidence supports an iron transport model in which iron-loaded transferrin is taken up by receptor-mediated endocytosis at the luminal membrane of brain capillaries. The iron then dissociates from transferrin in endosomal compartments and is transcytosed by unknown mechanisms, while the transferrin is retroendocytosed.
Morphological and biochemical analyses of contractile proteins (actin, myosin, caldesmon and tropomyosin) in normal and transformed cells
Authors: Tanaka Junya , Watanabe Tatsuo , Nakamura Norimasa , Sobue Kenji 
Published: April 2021

ABSTRACT
The expression and intracellular distribution of four contractile proteins (actin, myosin, caldesmon and tropomyosin) in normal fibroblasts and their transformed counterparts by Rous or avian sarcoma virus were compared. By analyzing the isoformal expression of actin, caldesmon and tropomyosin using two-dimensional gel electrophoresis, only tropomyosin showed significant alteration in its isoformal expression accompanied by transformation. Morphological study revealed that in normal cells, myosin, caldesmon and tropomyosin were distributed periodically along stress fibers, but were excluded from focal adhesions (adhesion plaques), at which stress fibers terminate. By contrast, the contractile proteins were concentrated within the protrusions of the ventral cell surface of transformed cells, which are cell-adhesive structures with high motility (podosomes). Regional analysis indicated that the contractile proteins do not show diffuse distribution within podosomes. Myosin, some caldesmon and tropomyosin in association with F-actin were localized in the region surrounding the core domains of podosomes. A major part of the caldesmon was, however, located in the core domain with short F-actin bundles. In order to compare the stability and the molecular organization of stress fibers with that of the short F-actin bundles within podosomes, the dorsal plasma membranes of the cells were removed by lysis and squirting. Then, the ruptured cells were treated with various buffers containing high salt, ATP or Ca2 /calmodulin. Myosin, caldesmon and tropomyosin were strongly associated with stress fibers of the ruptured normal fibroblasts even in a buffer containing high salt or Ca2 /calmodulin. On the other hand, myosin and tropomyosin within podosomes were easily extracted by lysis and squirting. And, the remaining caldesmon in podosomes was separated from the short F-actin bundles with high salt or Ca2 /calmodulin buffer. The present findings suggest that the high motility of podosomes from transformed cells is based on the actomyosin system, and that the stable adherence of focal adhesions of normal cells is due to a lack of this system. The accumulation of contractile proteins and their dynamic association within podosomes might be the cause of the short half-life of the structures. In relation to its localization in the core domain of podosomes without myosin and tropomyosin, the function of caldesmon has been discussed.
Microtubule organization during the cell cycle of the primitive eukaryote dinoflagellate Crypthecodinium cohnii
Authors: Perret Eric , Davoust Jean , Albert Marie , Besseau Laurence , Soyer-Gobillard Marie-Odile 
Published: April 2021

ABSTRACT
The complete microtubular system of the dinoflagellate Crypthecodinium cohnii Biecheler is described, as seen by confocal laser scanning fluorescence microscopy and labelling with anti-β-tubulin antibody. This technique allowed us to observe the organization of the subcortical and internal cytoskeletons and the mitotic microtubular system, and their changes during the cell cycle. These observations are compared with those made in cryosections by light microscopy and in fast-freeze-fixed, cryosubstituted cells by electron microscopy. We show the organization of the cortical microtubules, and in particular of the thick microtubular bundles arranged as a three-pronged fork from which they seem to emanate. This fork emerges from a peculiar cytoplasmic zone at the pole of the cell and is in contact with the region of the kinetosomes, at the cingulum. During the G1 phase, only a single, radial microtubular bundle (a “desmose”) is observable in the inner part of the cytoplasm. One of its ends is near the flagellar bases and the other end is close to the nucleus in the centrosome region. During the S phase, the flagella drop off, the cell encysts and the kinetosomes duplicate. In mitosis, the cortical microtubules and the intracytoplasmic microtubular bundles do not depolymerize. The microtubular fork, desmose and centrosome double and migrate, while the divided kinetosomes stay in the same place. Later, the centrosomes organize the extranuclear spindle, which is connected to the kinetosome region by the microtubular desmose. The convergent end of the threepronged fork seems to be in contact with the centrosome region. In early and mid-prophase, thick microtubular bundles pass through the nucleus in cytoplasmic channels and converge towards the two poles. Asters were never seen at the spindle poles. The channels and microtubular bundles in the spindle double in number during late prophase and lengthen in early anaphase. The spindle bundles diverge in late anaphase, extend to very near the plasma membrane and depolymerize during telophase. The cleavage furrow in which tubulin and actin are characterized appears in anaphase, formed by invagination of plasma membrane in the kinetosome region. The structure and rearrangements of the Crypthecodinium cohnii microtubular system are compared with those of other dinoflagellates and protists and of higher eukaryotes.
Characterization of a novel 63 kDa membrane protein : Implications for the organization of the ER-to-Golgi pathway
Authors: Schweizer Anja , Ericsson Maria , Bächi Thomas , Griffiths Gareth , Hauri Hans-Peter 
Published: April 2021

ABSTRACT
Owing to the lack of appropriate markers the structural organization of the ER-to-Golgi pathway and the dynamics of its membrane elements have been elusive. To elucidate this organization we have taken a monoclonal antibody (mAb) approach. A mAb against a novel 63 kDa membrane protein (p63) was produced that identifies a large tubular network of smooth membranes in the cytoplasm of primate cells. The distribution of p63 overlaps with the ER-Golgi intermediate compartment, defined by a previously described 53 kDa marker protein (here termed ERGIC-53), as visualized by confocal laser scanning immunofluorescence microscopy and immunoelectron microscopy. The p63 compartment mediates protein transport from the ER to Golgi apparatus, as indicated by partial colocalization of p63 and vesicular stomatitis virus G protein in Vero cells cultured at 15°C. Low temperatures and brefeldin A had little effect on the cellular distribution of p63, suggesting that this novel marker is a stably anchored resident protein of these pre-Golgi membranes. p63 and ERGIC-53 were enriched to a similar degree by the same subcellular fractionation procedure. These findings demonstrate an unanticipated complexity of the ER-Golgi interface and suggest that the ER-Golgi intermediate compartment defined by ERGIC-53 may be part of a greater network of smooth membranes.
Lipogenic enzyme and apoprotein messenger RNAs in long-term primary culture of chicken hepatocytes
Authors: Douaire M. , Belloir B. , Guillemot J.-C. , Fraslin J.-M. , Langlois P. , Mallard J. 
Published: April 2021

ABSTRACT
Hepatocytes isolated from 9-week-old chickens were cultured in a serum-free, hormonally defined medium. Relative amounts of mRNAs coding for lipogenic enzymes (acetyl-CoA carboxylase, fatty acid synthase, 9 desaturase, malic enzyme) and apoproteins (apoprotein A1 and apoprotein B) were determined until the 12th day. β-actin and albumin mRNA, as well as albumin secretion, were also assessed.
Cellular metabolic activity appeared to be very low for the first days of culture, but increased after the 7th day. All the mRNAs studied, except for that of malic enzyme, were present from this time throughout the culture lifespan. The biological significance of the observed results and the relevance of this chicken hepatocyte culture system for long-term metabolic and genetic studies are discussed.
Rapid de novo formation of gap junctions between insect hemocytes in vitro: a freeze-fracture, dye-transfer and patch-clamp study
Authors: Churchill Dennis , Coodin Sheppy , Shivers Richard R. , Caveney Stanley 
Published: April 2021

ABSTRACT
Gap junctions form between insect hemocytes (blood cells) when they encapsulate foreign objects in the hemocoel (body cavity). In this study we show that hemocytes from cockroach ( Periplaneta americana) form gap-junctions rapidly in vitro. Freeze-fracture replicas of hemocyte aggregates fixed 5 minutes after bleeding contain gap-junctional plaques. Dye passage was detected between carboxyfluorescein diacetate-labelled and unlabelled hemocytes within 3 minutes of bleeding, when the cells made contact as they flattened rapidly onto coverslips. When double whole-cell voltage-clamp was used to measure gap-junction formation between cells which were pushed together, electrical coupling was detected within one second of cell-cell contact. To prevent extensive flattening, cells were plated onto lipophorin-coated coverslips. Junctional conductance increased in staircase fashion with steps corresponding to an average single channel conductance of 345 pS. Assuming all channels to have this conductance, the maximal accretion rate of channels to the growing junction was one channel per second. Junctional currents and dye-coupling were detected in the absence of Ca2+, indicating that involvement of Ca2+-dependent adhesion molecules is not a prerequisite for gap-junction formation in hemocytes. Hemocytes from distantly related insects (cockroach and moth) form functional gap junctions with each other, suggesting sequence homology among gap-junction proteins in insects. The function of rapid gap-junction formation between hemocytes during encapsulation and wound healing in vivo are discussed.