bluuebunny/crossref_metadata_2025 huggingface dataset:
Organ maintenance of human sebaceous glands: in vitro effects of 13-cis retinoic acid and testosterone
Authors: Ridden John , Ferguson D. , Kealey Terence
Published: April 2021

Human sebaceous glands were isolated by shearing, and maintained for 7 days either on defined medium, on medium supplemented with 3/μM-tes-tosterone or on medium supplemented with both 3 μM-testosterone and 1 μM-13-cis retinoic acid.

Freshly isolated glands retained their in vivo morphology. On maintenance, the glands retained their freshly isolated rates of cell division, but the sebocytes showed increased keratinization and there was multilayering of the peripheral undifferentiated cells. However, glands maintained in the presence of 1 μM-13-cis retinoic acid showed very little luminal keratinization and only a small degree of multilayering.

On autoradiography, freshly isolated glands retained their in vivo pattern of [methyl-3H] thymidine incorporation. Similar patterns were seen when glands were maintained for 7 days with or without testosterone. However, in the presence of both testosterone and 13-cis retinoic acid there was only slight graining.

Following 7 days maintenance the rate of lipo-genesis fell significantly. This was partially reversed by testosterone, but further inhibited by 13-cis retinoic acid. The patterns of lipids that are synthesised after a week’s maintenance are very similar to those seen in freshly isolated glands, except that the squalene:cholesterol ratio is reversibly regulated by 3 μM-testosterone and 1 μM-reti-noic acid.

Protein synthesis was maintained at the same rates as for freshly isolated glands under all conditions of maintenance. Whereas DNA synthetic rates were maintained in the presence of testosterone, they were significantly inhibited by 13-cis retinoic acid.

Glandular wet weights were retained under all conditions of maintenance, except that they were significantly reduced by 13-cis retinoic acid.

This study shows that human sebocytes continue to divide on organ maintenance, but that they do not differentiate fully. However, this provides the first demonstration that 13-cis-retinoic acid acts on human sebaceous glands directly, reducing the rate of cell division and the rate of lipogenesis, which shows that the maintained human sebaceous gland might provide a useful model for studying the effect of 13-cis retinoic acid on human sebocytes.
Steps in the morphogenesis of a polarized epithelium: II. disassembly and assembly of plasma membrane domains during reversal of epithelial cell polarity in multicellular epithelial (MDCK) cysts
Authors: Wang Allan Z. , Ojakian George K. , Nelson W. James
Published: April 2021

A fundamental aspect in the morphogenesis of a polarized epithelium is the formation of structurally and functionally distinct apical and basal-lateral domains of the plasma membrane. The formation of these membrane domains involves the accumulation of domain-specific proteins and removal of incorrectly localized proteins. The mechanisms involved in these processes are not well understood. We have approached this problem by detailed analysis of the distribution and fate of proteins specific for different membrane domains during reversal of epithelial polarity. In the preceding paper we showed that MDCK cells form multicellular cysts comprising a closed monolayer of polarized cells. The orientation of cell polarity depends upon whether cysts are formed in suspension culture or in a collagen gel. Here, we show that, when fully developed cysts formed in suspension culture are placed in a collagen gel, polarity is rapidly reversed without cell dissociation. We show that during the process of polarity reversal, plasma membrane domains are disassembled by uptake of proteins into cytoplasmic vesicles, followed by protein degradation that probably occurs in lysosomes. The disassembly and assembly of the apical and the basal-lateral membrane domains occur in a sequential order with different kinetics. Our results provide further insights into the establishment of protein specificity of plasma membrane domains in polarized cells.
Requirement for two different cell-binding domains in fibronectin for neurite extension of neuronal derivative cells
Authors: Lewandowska Kristine , Balza Enrica , Zardi Luciano , Culp Lloyd A.
Published: April 2021

Some neuron-derived cells, such as neuroblastoma cells, adhere and extend neurites on fibronectin (FN) substrata by processes that can be independent of binding to the Arg-Gly-Asp-Ser sequence (RGDS in FN) and independent of proteoglycan/ganglioside-binding activities of FN. Proteolytic fragments of various FNs have been used in this study to map a new adhesion-promoting domain in FNs that may be neural cell-specific. A thermolysin-generated fragment of human plasma FN (Fl 10 containing the RGDS domain) or the analagous fragment from transformed human cell FN (F120, also containing the alternately spliced extra domainb[EDb]) facilitate RGDS-independent adherence and neurite extension of human neuroblastoma cells and an Fll hybrid neuronal line (by fusion of mouse neuroblastoma cells with rat dorsal root ganglion neurons) as effectively as adherence and neurite extension on intact FN. Since neither F110 nor F120 contains sequences from the alternately spliced IIICS region of FN, neurite-promoting activity in these fragments cannot be ascribed to a recently discovered cell-binding domain in this region. Furthermore, F120 could be cleaved into two subfragments retaining virtually all the sequence of the parent fragment: F35 from the C terminus of F120 containing the RGDS domain, and F90 from the N terminus containing most of the EDb region bordering the thermolysin cleavage site. These neuronal cells could adhere but not extend neurites on substrata coated with either F35 or F90 alone while 3T3 cells could adhere only on F35. Mixtures of F35 and F90 on substrata could reconstitute some, but not nearly all, of the neurite-promoting activity of F120. Therefore, these data identify a new cell-binding domain in common sequences of FNs on the N-terminal side of EDb and demonstrate cooperativity between this RGDS-in-dependent domain and the RGDS-dependent domain for maximal differentiation of these neuron-derived cells. Several possibilities for a receptor directed to this new domain are discussed.
Cytokeratin expression in rat mesothelial cells in vitro is controlled by the extracellular matrix
Authors: Mackay Alastair M. , Tracy Russell P. , Craighead John E.
Published: April 2021

Rat mesothelial cells co-express vimentin and the simple epithelial cytokeratins. While cytokeratins predominate in situ, under most culture conditions vimentin is the major intermediate filament protein of the cells. This loss of cytokeratin production upon culture can be partly prevented by growing mesothelial cells on a basement membrane matrix. However, the basement membrane-promoted persistence of cytokeratin synthesis is not accompanied by expression of cytokeratin G (no. 19), the major acidic cytokeratin of mesothelium in vivo.

While cells grown on plastic establish a prominent juxtanuclear assemblage of tonofilaments, those cultured on basement membrane exhibit cytokeratin filaments which are distributed throughout the cytoplasm and attach to neighboring cells at the plasma membrane. This latter pattern resembles that seen in the intact mesothelium.

Intermediate filaments are markers of cellular differentiation, but their roles are obscure. The response of cultured mesothelial cells to different growth substrata supports the hypothesis that intermediate filament synthesis is influenced by cellular contact with the extracellular matrix.
Nerve branching is induced and oriented by a small applied electric field
Authors: McCaig C. D.
Published: April 2021

Nerve branching is controlled by intrinsic and extrinsic cues, one of which may be a small applied electric field. Lateral processes were induced by passing current through a micropipette placed at 90° to the shaft of a developing nerve. The appearance of processes was a polarised event with a large majority arising from the cathodal facing side of nerves. Whilst an electric field alone may promote branching, the presence of dimethyl sulfoxide (DMSO) or the ganglioside GM! enhanced branching of developing nerves. It is likely that an applied electric field promotes microtubule disassembly locally along the neurite shaft and that this can lead to a polarised rearrangement of the neuronal cytoskeleton. It is suggested that the use of an applied electric field in conjunction with these pharmacological agents might enhance nerve regeneration in vivo.
C-cam (cell-CAM 105) is an adhesive cell surface glycoprotein with homophilic binding properties
Authors: tingström Anders , blikstad Ingrid , aurivillius Magnus , Öbrink Björn
Published: April 2021

C-CAM (Cell-CAM 105) is a cell surface glycoprotein that is involved in cell-cell adhesion of rat hepatocytes in vitro. To elucidate the adhesion mechanism the binding properties of purified C-CAM were investigated. Using proteins immobilized on nitrocellulose it was found that radiolabeled C-CAM bound to C-CAM but not to a variety of other proteins. Partitioning in Triton X-114 showed that C-CAM has hydrophobic properties. In accordance with this, C-CAM was effectively incorporated into phosphatidylcholine liposomes by dialysis from octylglucoside-containing solutions. The C-CAM-containing liposomes bound specifically to isolated hepatocytes. This binding was blocked by Fab fragments of antiC-CAM antibodies. Furthermore, preincubation of hepatocytes with anti-C-CAM antibodies followed by washing of the cells blocked binding of C-CAM-containing liposomes. At increasing C-CAM contents in the reconstituted liposomes a marked self-aggregation of the liposomes occurred. This aggregation was blocked by Fab fragments of anti-C-CAM antibodies and by alkaline pH. After neutralization a rapid reaggregation occurred. Neither C-CAM binding to C-CAM immobilized on nitrocellulose nor C-CAM-liposome aggregation required calcium ions. Liposomes reconstituted with C-CAM-depleted membrane glycoproteins did not self-aggregate or bind to hepatocytes. Thus, it is concluded that C-CAM can bind specifically to C-CAM in a homophilic binding reaction that does not require calcium. Accordingly, C-CAM has the potential of directly mediating cell-cell adhesion via C-CAM-C-CAM binding between adjacent cells.
Distribution of tubulin and actin through the cell division cycle of the fission yeast Schizosaccharomyces japonicus var. Versatilis: a comparison with Schizosaccharomyces pombe
Authors: Alfa Caroline E. , Hyams Jeremy S.
Published: April 2021

Changes in the distribution of microtubules and F-actin through the cell division cycle of the fission yeast Schizosaccharomyces japonicus var. versatilis were investigated by fluorescence microscopy. The fluorescence images obtained with S. japonicus were markedly superior to those previously reported for S. pombe and revealed new details of cytoskeletal organization in this important genus. As in S. pombe, F-actin in S. japonicus was present as a concentration of ‘dots’ at the growing poles of interphase cells and as a filamentous equatorial ring directing the deposition of the cytokinetic septum. The transition between these two states occurred at late anaphase, in contrast to the situation in S. pombe where the appearance of the equatorial actin ring is tightly coupled to the early events of mitosis. During the course of cytokinesis in S. japonicus the actin ring constricted and broadened, suggesting that it is contractile. Microtubule organization in S. japonicus also revealed interesting differences from S. pombe. Whereas in S. pombe cytoplasmic microtubules are reinitiated from a pair of microtubule organizing centres (MTOCs) at the cell equator, in S. japonicus they arise by extensive microtubule growth from the spindle poles. Western blots of cell extracts enriched for tubulin by DEAE-Sephadex chromatography showed that, like S. pombe, S. japonicus contains two a-tubulins and a single β-tubulin. Whilst the al- and fi-tubulins from the two species comigrated on onedimensional polyacrylamide gels, the a2 species were electrophoretically distinct Although fundamental differences clearly exist between the two species, S. japonicus could prove to be a useful tool in basic studies of fission yeast cell biology.
Increased gap junction assembly between cultured cells upon cholesterol supplementation
Authors: Meyer Rita , Malewicz Barbara , Baumann Wolfgang J. , Johnson Ross G.
Published: April 2021

Novikoff hepatoma cells provide an excellent model system for the study of gap junction assembly, a process that could be influenced by lipids and other factors at numerous points. Since it is possible to alter the cellular levels of cholesterol in these cells, it was added to the cells in serum-supplemented medium and changes in gap junction assembly were evaluated. Cells were dissociated and reaggregated following exposure to a range of cholesterol concentrations for 24 h. A five- to sixfold increase in the number of aggregated gap junction particles and a 50% increase in cellular cholesterol content were observed with 20 μM added cholesterol. A 1-h exposure to added cholesterol, during cell reaggregation, resulted in a fourfold increase in the number of aggregated gap junction particles, demonstrating that the effect was rapid. The number of aggregated gap junction particles and formation plaque areas were used as measures of junction assembly and assayed by quantitative freeze-fracture and electron microscopy. Junctional permeabilities were evaluated by means of dye transfer times following the intracellular microinjection of Lucifer Yellow. Increased dye transfer was observed between cholesterol-treated cells, which suggested that the increase in assembly was accompanied by an increase in junction permeability. Cells were treated with cycloheximide (100μgml−1) and actinomycin D (10 μgml−1) to determine whether protein and RNA syntheses were involved in the enhanced gap junction assembly. Cycloheximide but not actinomycin D blocked the increased junction assembly observed with added cholesterol. These results suggested that protein synthesis, but not RNA synthesis, is necessary for the increased gap junction formation observed. Thus, cholesterol added in the presence of serum-supplemented medium resulted in a dramatic and rapid increase in the assembly of functional gap junctions between Novikoff cells.
Growth factors for human fibroblasts in the solute remaining after clot formation
Authors: Gray Andrew J. , Reeves Jack T. , Harrison Nicholas K. , Winlove Peter , Laurent Geoffrey J.
Published: April 2021

Fibroblasts adhere to, and readily grow into, fibrin clots that form as a result of the cleavage of fibrinogen by thrombin. Subsequent fibroblast replication is believed to be stimulated by mitogens released by entrapped platelets, such as platelet-derived growth factor. We suggest that the supernatant remaining after the fibrinogen-thrombin reaction could stimulate fibroblast replication, even in the absence of other blood components. To examine this hypothesis we expressed liquid from a fibrin clot and measured its mitogenic activity on human lung fibroblasts, in serum-free conditions, using a colormetric assay based on uptake and subsequent release of Methylene Blue. The clot supernatant caused a mitogenic response of 51±6 % above control and was equivalent to about half that elicited by medium containing 10 % newborn calf serum. On their own, both thrombin and fibrinopeptides A and B (small molecular weight cleavage products released from fibrinogen) showed some mitogenic activity, but there was also activity in higher molecular weight cleavage products, suggesting the presence of uncharacterised mitogens. It is proposed that these agents may play important roles in wound healing and diseases associated with vascular leakage and fibrosis, by stimulating fibroblast replication.
Spectrin, actin and the structure of the cortical lattice in mammalian cochlear outer hair cells
Authors: Holley M. C. , Ashmore J. F.
Published: April 2021

Mammalian cochlear outer hair cells generate high-frequency forces in response to electrical stimulation. Force generation occurs in the lateral cortex of the cell, which includes the plasma membrane, a two-dimensional ‘cortical lattice’ of filamentous protein, and a multi-layered membrane system, the lateral cistemae. The cortical lattice is composed of relatively long filaments, 6.7 nm in diameter, which are wound circumferentially about the cell. These filaments are spaced about 42 nm apart and are cross-linked by a second type of filament 3.2 nm in diameter approximately aligned with the longitudinal axis of the cell. The cortical lattice is the only cortical structure that remains after the cell is fully extracted in the detergent Triton X-100 and high-salt solution. It retains the original cylindrical shape of the cell and is reversibly deformable. Antibodies raised against chicken gizzard actin, human blood spectrin and pig brain spectrin all react positively with the extracted lattice viewed using immunofluorescence. Three protein subunits identified in the organ of Corti have approximate molecular weights of 220, 235 and 240K (K= 103Mr) and react with the spectrin antibodies. A structural model of the lattice is proposed in which the circumferential filaments are composed of actin and the cross-links of spectrin. The model can account for the unusual cylindrical shape of outer hair cells and suggests a mechanism of force generation based upon the elastic and electrostatic properties of spectrin.
Import of proteins into chloroplasts
Authors: Flügge Ulf-Ingo
Published: April 2021

Chloroplasts are the sites of photosynthesis in eukaryotic plants and are able to harvest solar energy for the synthesis of carbon skeletons. They, like mitochondria, are enclosed by two membranes; the outer and the inner envelope. The latter is the actual permeability barrier between the cytosol and the chloroplast stroma and the site of different metabolite translocators that coordinate the metabolism between compartments (Heldt and Flügge, 1987). Distinct from the envelope membranes are the energy-transducing thylakoid membranes that are located within the chloroplasts, and which enclose the thylakoid lumen. Stroma and thylakoid proteins each account for about 50% of the total chloroplast protein, whereas the two envelope membranes represent less than 1%. In spite of the chloroplasts’ semi-autonomy the majority of the chloroplasts proteins and also those of other cell compartments are coded for in the nucleus. These nuclear-coded proteins are synthesized in the cytosol, normally with N-terminal extensions called transit peptides, and are subsequently transported into the chloroplasts where they are processed to their mature sizes by specific proteases. Mechanisms must therefore exist to ensure that these proteins are (1) correctly targeted to chloroplasts and (2) subsequently localized into the correct chloroplast compartment: namely, the outer or inner envelope membrane, the intermembrane space, the chloroplast stroma, the thylakoid membrane or the thylakoid lumen. Major aspects of protein transport have been reviewed quite recently (Schmidt and Mishkind, 1986; Lubben et al. 1988; Keegstra et al. 1989). This Commentary will focus on the basic phenomena of this topic and will also present recent findings that extend the view of the reviews cited above.
The monovalent ionophore monensin maintains the nuclear localization of the human stress protein hsp28 during heat shock recovery
Authors: Arrigo AndrÉ-Patrick
Published: April 2021

In HeLa cells exposed to supra-optimal temperatures, the a-crystallin-related stress protein hsp28 is reversibly redistributed inside the nucleus and increases its level of phosphorylation and aggregation. Here, I show that, at normal temperature after a heat stress, the sodium ionophore monensin maintains the nuclear localization of hsp28 without impairing the dephosphorylation of this protein. This phenomenon is not due to a prolongation, by monensin, of the synthesis of the heat-shock proteins after the heat stress. In contrast, the potassium ionophore nonactin induces only a weak alteration in the hsp28 locale, while the calcium ionophore A23187 and the uncoupler of oxidative phosphorylation FCCP have no effect. Following the removal of monensin 15 h after the heat stress, a further incubation of the cells for at least 36 h is necessary in order to observe a redistribution of hsp28 into the cytoplasm. A large fraction of hsp28 is then observed as dense excretion granules. In control cells kept at normal temperature, monensin, like nonactin, A23187 and FCCP, does not induce the redistribution of hsp28 inside the nucleus. Taken together, these results suggest that the disruption of the Na+ active transport by monensin probably inhibits the redistribution of hsp28 in the cytoplasm after heat shock.
Ribonuclease H(70) is a component of the yeast nuclear scaffold
Authors: Karwan Robert M. , Laroche Thierry , Wintersberger Ulrike , Gasser Susan M. , Binder Maximilian
Published: April 2021

We have used monospecific antibodies against three ribonuclease H enzymes of Saecharomyces cerevisiae to investigate their intracellular localization. Fractionation experiments, as well as immunocytochemical staining, revealed a predominantly cytoplasmic localization of the RNase H proteins of 42 000 and 70 000, Mr, whereas that of 55000 Mr showed equal distribution between nuclei and cytoplasm. The nuclear moiety of ribonuclease H(70) was found to be a part of the yeast nuclear scaffold, as investigated by immunoblotting and antibody inhibition experiments. The 42000 and 55000 Mr enzymes, on the other hand, are not scaffold-associated. We conclude that RNase H(70) is part of the nuclear substructure of yeast that was previously found to maintain specific interactions with yeast chromosomal origins of replication (ARS elements).
Mutations at the ASP locus of Drosophila lead to multiple free centrosomes in syncytial embryos, but restrict centrosome duplication in larval neuroblasts
Authors: Gonzalez C. , Saunders R. D. C. , Casal J. , Molina I. , Carmena M. , Ripoll P. , Glover D. M.
Published: April 2021

Mutations at abnormal spindle result in abnormally long and wavy microtubules in the meiotic spindles of males. Some of these spindles have a single pole and take the form of unopposed hemi-spindles. Unfertilised eggs produced by homozygous asp females may have either no nuclei, or a small number of large nuclei, consistent with there also being an effect upon female meiosis. Such eggs also display free centrosomes and independent arrays of microtubules. Embryos that have this phenotype are also present among the progeny of fertilised homozygous asp females, together with embryos that undergo varying degrees of aberrant morphogenesis, developing a variety of abnormal cuticle patterns. This latter category shows asynchronous mitoses prior to cellularisation, and has abnormal arrays of spindle microtubules. Such embryos can develop large areas that are either devoid of or have a reduced number of nuclei, in which there are centrosomes that have dissociated from the mitotic spindles. Neuroblasts in the brains of homozygous asp larvae display a high mitotic index, and have condensed chromosomes aligned as if blocked at metaphase. Immunostaining reveals that many cells contain a single centrosome connected to the metaphase chromosomes by microtubules in a hemi-spindle-like structure.
Neurofilament proteins are co-expressed with desmin in heart conduction system myocytes
Authors: Vitadello Maurizio , Matteoli Michela , Gorza Luisa
Published: April 2021

We have recently shown that specialized myocytes of the rabbit heart express a cytoskeletal protein similar to the M subunit of neurofilaments (NF). Since this result was obtained using a single anti-NF-M monoclonal antibody, we tested on conduction myocytes a panel of five anti-NF antibodies, specific for each of the three NF subunits and for phosphorylated and non-phosphorylated epitopes. Two antibodies, one specific for the L subunit and one for phosphorylated M subunit of NF, reacted with specialized myocytes in immunohistochemistry. In immunoblots on conduction tissue homogenates the two antibodies recognized two polypeptides with electrophoretic mobility and solubility properties identical to those of NF-L and NF-M in the sciatic nerve.

The subcellular distribution of NF immunoreactivity in specialized myocytes was very similar to desmin localization; namely, it was distributed on large filamentous bundles and on fine filaments localized transversely at the level of the Z line. At the ultrastructural level, immunoreactive filaments were localized in the intermyofibrillar space and connected myofibrils with mitochondria. Co-expression of NF proteins and desmin was also observed in vitro in a minor population of cardiac myocytes cultured from embryonic rabbit heart. In most cases NF immunoreactivity co-localized with desmin, especially where filaments were well organized, but in some cells anti-NF and anti-desmin antibodies labelled different filamentous structures.

These results indicate that NF proteins are structural components of the cytoskeleton of specialized myocytes and show a subcellular distribution very similar to desmin. Such a composition of intermediate filaments indicates that in these cardiac cells muscle differentiation is compatible with the expression of neuronal proteins.
Adsorptive endocytosis and membrane recycling by cultured primary bovine brain microvessel endothelial cell monolayers
Authors: Raub Thomas J. , Audus Kenneth L.
Published: April 2021

The dynamics of membrane recycling were examined in primary cultures of brain microvessel endothelial cells (BMECs). Because the BMEC surface was dominated by galactosylated glycoconjugates, ricin agglutinin (RCAI) was used as a tracer to follow the endocytosis and recycling of RCAI binding sites. These binding sites accounted for 75% of the iodinatable or most externally disposed plasma membrane proteins. Because greater than 90% of the RCAI that had bound to BMECs was removed by a brief, nontoxic treatment with galactose, the amounts and kinetics for internalization and efflux of [125I]RCAI were measured. Both endocytosis and efflux were energy dependent. By using pseudo-first-order kinetics, the t 1 values for RCAI binding, internalization and efflux were 5, 18 and 13–14 min, respectively. By comparing efflux with and without galactose present, we found that 60% of the RCAI binding sites that had been internalized were returned to the cell surface and reinternalized. Quantifying the distribution of gold-RCAI following internalization showed kinetics consistent with that obtained using radiolabeled RCAI. Both horseradish peroxidase (HRP) and gold-conjugated RCAI that had bound BMEC at 4 °C became localized within more caveolae within 2.5min of warming to 37°C to permit endocytosis. With time, RCAI appeared within endosomes and tubules and vesicles of which some were located in the trans-Golgi network (TGN). The distribution of HRP-RC Al contrasted with that of free HRP, which was not routed to the TGN. The absence of RCAI conjugates in association with the basolateral membrane domain suggested the presence of functional tight junctions and maintenance of polarity throughout the duration of these experiments. These results showed that membrane recycling was more extensive and much slower than fluid-phase endocytosis in cultured BMECs. Moreover, we found that endocytosis of membrane by BMECs in culture was similar to that reported for brain endothelium in vivo in that a fraction of the cell surface membrane was routed to the TGN.
Laminin potentiates differentiation of PCC4uva embryonal carcinoma into neurons
Authors: Sweeney T. M. , Ogle Roy C. , Little C. D.
Published: April 2021

The embryonal carcinoma PCC4uva differentiates into neurons in response to treatment with retinoic acid and dbcAMP. We used this in vitro model system to study the effects of laminin on early neural differentiation. Laminin substrata markedly potentiate neural differentiation of retinoic acid and dbcAMP-treated cultures. Only laminin induced more rapid neural cell body clustering, neurite growth and neurite fasciculation as compared to type IV collagen, type I collagen, and fibronectin substrata. Exogenous laminin substrata promoted greater cell attachment, cellular spreading and growth to confluence than type IV collagen, type I collagen, fibronectin and glass substrata. Laminin-induced effects were inhibited by addition of laminin antibodies or the synthetic laminin-derived peptide De-Gly-Ser-Arg-NH2 (YIGSR-NH2). Treatment with YIGSR-NH2also inhibited neural differentiation in the absence of exogenous laminin substrata, whereas synthetic peptides containing the RGD sequence and a control peptide YIGSK-NH2 showed no inhibitory effects. These results are consistent with the hypothesis that specific interactions between an early differentiating cell population(s) and extracellular laminin are required during neural differentiation.
Blood platelet formation in vitro. The role of the cytoskeleton in megakaryocyte fragmentation
Authors: Tablin Fern , Castro Marlene , Leven Robert M.
Published: April 2021

We have developed a unique in vitro model that promotes differentiation of megakaryocytes into platelets. When megakaryocytes isolated from guinea pig bone marrow were cultured on hydrated rat tail collagen gels, cells spontaneously formed elongated, beaded processes that fragmented to yield cytoplasmic pieces with the same size and internal composition as individual platelets. Addition of nocodazole at the initiation of cultures blocked process formation, while addition of nocodazole to cells with previously established processes resulted in their retraction. The addition of taxol to cultures resulted in abnormally thick processes that were tightly adherent to the underlying substratum, and did not bead or fragment Cytochalasin D accelerated process formation and fragmentation of megakaryocytes cultured on collagen gels by twofold. On the basis of these results, we propose a model for platelet formation in culture that involves the following steps: adherence of megakaryocytes to the underlying extracellular matrix; dilation of the demarcation membrane system and breakdown of the actin-rich peripheral zone; microtubule-based extension of pseudopodia, which are no longer adherent to the substratum; and fragmentation into platelets by the coalescence and fusion of demarcation membrane vesicles with the plasma membrane. We feel that this distinctive culture system closely approximates thrombocytopoiesis in vivo, thus allowing detailed elucidation of this important process.
Localization of platelet-derived growth factor (PDGF) in CHO cells transfected with PDGF A- or B-chain cDNA: Retention of PDGF-BB in the endoplasmic reticulum and Golgi complex
Authors: Thyberg Johan , Östman Arne , Bäckström Gudrun , Westermark Bengt , Heldin Carl-Henrik
Published: April 2021

Platelet-derived growth factor (PDGF) is a powerful mitogen for connective tissue cells. It is made up of two polypeptide chains (A and B) and exists in three dimeric forms (AA, AB, and BB). Transfection experiments have indicated that PDGF-AA and -AB are secreted as 30×103Mr products, whereas PDGF-BB is processed into a 24×103Afr product and remains associated with the cells. Here, CHO cells were transfected with PDGF B- or A-chain cDNA and the intracellular distributions of the respective gene products were compared by indirect immunofluorescence and immunoelectron microscopy, using primary antibodies specific for PDGF B- and A-chain homodimers. PDGF-BB was most conspicuous in stacked Golgi cisternae. It was also found in the endoplasmic reticulum and in lysosomes. Upon treatment of the cells with the microtubule-disruptive drug nocodazole, the Golgi complex was broken up and its stacks of cisternae were dispersed throughout the cytoplasm together with clusters of lysosomes. After this structural disorganization, the concentration of PDGF-BB to the Golgi stacks was even more prominent than before. Weak reactivity for PDGF-AA was detected in the endoplasmic reticulum and groups of vacuoles, both in control and nocodazole-treated cells, whereas Golgi stacks and lysosomes only seldom were positive. The observations suggest that PDGF-BB is processed and retained within the endoplasmic reticulum and Golgi complex. Eventually, it may also be transferred to lysosomes for degradation. In contrast, PDGF-AA is likely to follow a pathway for bulk flow, including rapid passage through the endoplasmic reticulum and Golgi complex, package in secretory vacuoles, and extracellular release by exocytosis.
Characterization of the ZO-1 protein in endothelial and other cell lines
Authors: Li Changxin , Poznansky Mark J.
Published: April 2021

A high molecular weight tight junction-associated protein, ZO-1, has been demonstrated in liver (hepatocytes) and in both epithelium and endothelium. We carried out studies to examine the presence of the protein in vascular endothelial cell cultures and several other types of cultured cells, and the relationship between the ZO-1 protein content and confluency of endothelial cell monlayers. Immunofluorescence labelling of endothelial monolayers and two types of epithelial monolayers, IEC-6 and MDCK, with monoclonal antibody against ZO-1 protein localized the protein to the cell peripheries. Its association with the cell periphery only occurred when cells had contact with one another as demonstrated in endothelial cells. We have been able to show a positive correlation between the ZO-1 content of the cells and the extent of monolayer confluency in the endothelial cells by immunoblotting. The protein is much less expressed in nonconfluent endothelial cell monolayers and totally absent from mouse myeloma cultures. The presence and confluence-related expression of the protein in endothelium give support to the hypothesis that tight junctions exist in confluent endothelial cells and that the ZO-1 protein is expressed under the conditions where tight junction interactions occur.
Assessing the differentiation state of cultured bovine urothelial cells: elevated synthesis of stratification-related K5 and K6 keratins and persistent expression of uroplakin I
Authors: Surya Babu , Yu Jun , Manabe Motomu , Sun Tung-Tien
Published: April 2021

Although significant progress has recently been made in culturing mammalian urothelial cells, relatively little is known about their biochemical differentiation. In this paper, we assessed the differentiation state of cultured bovine urothelial cells by analyzing their keratins and a cell surface marker, uroplakin I. Urothelial cells were serially cultured either in a serum-free medium, or in a serumcontaining medium in the presence of 3T3 feeder cells, with similar results. Despite their stratified appearance, both normal urothelium and cultured urothelial cells synthesize mainly K8, K18 and K19, keratins that are typically seen in simple epithelia. However, cultured urothelial cells synthesize a greatly increased amount of K5 and K6 keratins, which are usually expressed by stratified epithelia but present only in trace amounts in normal urothelium. These data indicate that, as far as keratin synthesis is concerned, cultured urothelial cells undergo an altered pattern of differentiation towards a more ‘stratified phenotype’; this unusual finding has interesting implications for urothelial evolution. In the meantime, many superficial cells in cultured urothelial colonies make uroplakin I, a 27 × 103Mr protein subunit of the asymmetrical unit membrane (AUM) characteristic of urothelial (superficial) umbrella cells. These results indicate that cultured urothelial cells undergo, at least in part, AUM biogenesis. Cultured urothelial cells thus provide a useful experimental model system for studying certain early steps of AUM formation.
Human hair growth in vitro
Authors: Philpott Michael P. , Green Martin R. , Kealey Terence
Published: April 2021

We report for the first time the successful maintenance and growth of human hair follicles in vitro. Human anagen hair follicles were isolated by microdissection from human scalp skin. Isolation of the hair follicles was achieved by cutting the follicle at the dermo-subcutaneous fat interface using a scalpel blade. Intact hair follicles were then removed from the fat using watchmakers’ forceps.

Isolated hair follicles maintained free-floating in supplemented Williams E medium in individual wells of 24-well multiwell plates showed a significant increase in length over 4 days. The increase in length was seen to be attributed to the production of a keratinised hair shaft, and was not associated with the loss of hair follicle morphology. [methyl-3H]thymidine autoradiography confirmed that in vitro the in vivo pattern of DNA synthesis was maintained; furthermore, [35S]methionine labelling of keratins showed that their patterns of synthesis did not change with maintenance.

The importance of this model to hair follicle biology is further demonstrated by the observations that TGF-βl has a negative growth-regulatory effect on hair follicles in vitro and that EGF mimics the in vivo depilatory effects that have been reported in sheep and mice.
Mitochondrial growth and DNA synthesis occur in the absence of nuclear DNA replication in fission yeast
Authors: Sazer Shelley , Sherwood Steven W.
Published: April 2021

Cell growth and division require the doubling of cellular constituents followed by their equal distribution to the two daughter cells. Within a growing population, the ratio of mitochondrial to cellular volume is maintained, as is the number of mitochondrial genomes per cell. The mechanisms responsible for coordinating nuclear and mitochondrial DNA synthesis, and for balancing increases in cell and mitochondrial size are not well understood. In studies of the fission yeast Schizosaccharomyces pombe we quantified cellular and mitochondrial DNA content by both Southern blot analysis and flow cytometry of cells stained with a variety of DNA-binding fluorochromes, which we show are able to detect nuclear and mitochondrial DNA with different efficiencies. In the conditional cell division cycle mutant cdclO, which is unable to initiate nuclear DNA synthesis, we found that there was an increase in the mitochondrial DNA content in the absence of nuclear DNA replication. This demonstrates that mitochondrial and nuclear DNA synthesis are not obligately linked. We also show that mitochondrial DNA replication is not required for the increase in mitochondrial size that occurs as cells elongate, although this results in a decrease in the ratio of mitochondrial DNA to mitochondrial volume.
Identification of a 40×103Mr centromere-associated protein in cultured mammalian cells
Authors: Balczon Ron , Accavitti Mary Ann , Brinkley B. R.
Published: April 2021

Monoclonal antibodies were raised against a complex of proteins that was purified following the crosslinking of tubulin to the centromeres of CHO chromosomes using Lomant’s reagent. One of the clones, hybridoma 32–9, produced antibodies that reacted with a 40×103Mr protein present in the crosslinked complex. Furthermore, immunoblot analysis demonstrated that the 40×103Mr antigen was present in various mammalian cell types from several different species. Indirect immunofluorescence using the antibody produced by clone 32–9 demonstrated that the 40 × 103Mr antigen was associated with both spindle and cytoplasmic microtubules. Ln addition, centromere/kinetochore staining was detected in metaphase-arrested cells, while staining of prekinetochores in interphase nuclei was not observed. Unlike microtubule-associated pro teins and microtubule-dependent ATPases, the 40 ×103 Mr protein did not copurify with microtubules when tubules were assembled from cellular homogenates using taxol and either GTP or GTP and AMP-PNP. Instead, the 40×103Mr protein remained associated with the insoluble cellular material. The 40×103Mr antigen could be released from the insoluble pelleted material by extraction with 1M NaCl. Once solubilized, the 40×103Mr protein was able to copurify with microtubules in assembly assays in vitro. This monoclonal antibody should serve as a valuable probe for studies of centromere/kinetochore structure and function.
Hydrostatic pressure influences histone mRNA
Authors: Symington Alison L. , Zimmerman Selma , Stein Janet , Stein Gary , Zimmerman Arthur M.
Published: April 2021

Exposure of HeLa S3 cells to high hydrostatic pressure (6.89 ×103 to 6.89 ×104 kPa: 1000 to 10000lbfin-2) reduced core and H1 histone mRNA levels as determined by hybridization to specific histone DNA probes. At 4.14×104kPa for 10 min core histone and H1 histone mRNA levels were reduced 32–38% and 56%, respectively. At 30 min post-decompression core mRNA levels returned to atmospheric control levels while H1 histone mRNA levels continued to be suppressed. Levels of macromolecular synthesis were monitored under hydrostatic pressure with radioactive precursors of RNA, DNA and protein. Macromolecular synthesis was shown to be suppressed in a dose-dependent manner with increasing magnitude and duration of pressure. To determine the influence of pressure on histone mRNA stability, actinomycin D (10 μg ml-1) was used to block RNA synthesis. Relative amounts of H4 and H1 mRNA were determined at atmospheric pressure and following treatment with actinomycin D (10μgml1), pressure (4.14×104kPa) and a combination of pressure and actinomycin D. This study shows that a synthesis component and a stability component are involved in the pressure-induced reduction of core histone mRNA. At 4.14×104kPa for 15 min, there was a 42% reduction in core histone mRNA of which approximately one third was due a suppression of transcription and two thirds to a loss of mRNA stability. The pressure-induced reduction in histone mRNA is attributed to the instability of endogenous histone mRNA and a reduction in transcription/processing of new histone mRNA.
Human hepatocellular carcinoma cell lines exhibit multidrug resistance unrelated to Mdr1 gene expression
Authors: Shen D.-W. , Lu Yuan-G. , Chin Khew-V. , Pastan I. , Gottesman M. M.
Published: April 2021

Multidrug resistance of human cancer cells may result from expression of P-glycoprotein, the product of the MDR1 gene, acting as an energy-dependent drug efflux pump. However, direct evidence that expression of the MDR1 gene contributes to the multidrug resistance of human liver carcinomas has not been established. In this study, we tested five cell lines derived from human hepatocellular carcinomas for sensitivity to a variety of drugs used widely as anticancer agents: these included vinblastine, doxorubicin, actinomycin D, mitomycin C, 5-fluorouracil, 6-mercaptopurine, melphalan, methotrexate, cj’s-pla- tinum and etoposide (VP-16). All five hepatoma cell lines were resistant at different levels to these chemicals compared to human KB cells. Although it has been demonstrated that resistance to vinblastine, colchicine, doxorubicin and actinomycin D in human multidrug-resistant cells is associated with overexpression of P-glycoprotein, very little expression of P-glycoprotein was found in these human hepatoma cells. Neither verapamil nor quinidine, inhibitors of the drug efflux pump, were able to overcome multidrug resistance in hepatoma cells. These results indicate that the multidrug resistance phenotype in human hepatocellular carcinoma cells cannot be attributed to expression of the MDR1 gene, but that novel mechanisms may account for the resistance of these cancer cells.
Immunological identification of a new 14×103Mr membrane-bound protein in Torpedo electric organ
Authors: Morel Nicolas , Brochier Guy , Synguelakis Monique , Salle Gildas Le Gal La
Published: April 2021

A series of monoclonal antibodies binding to different epitopes shared by a 14×103Mr membrane-bound polypeptide has been obtained. By indirect immunofluorescence, it was shown that the 14×103Mr antigen is present in various cell types in Torpedo electric organ and muscle, especially fibroblasts, capillary endothelial cells, axonal cuff cells and, to a lesser extent, Schwann cells. At the electron-microscope level, after immunogold labelling, the antigen was found associated with the external surface of the plasma membrane of these cells, with the exception of the axonal cuff cells where part of the labelling was intracellular. The possible biological role of this 14×103Mr protein is unknown but preliminary experiments suggest that this antigen has affinity for other Torpedo electric organ membrane proteins.
Induction of cell processes by local force
Authors: Margolis L. B. , Popov S. V.
Published: April 2021

A method of applying local mechanical force to the plasma membrane of mouse embryo fibroblasts is described. The force is generated by local treatment of cells with an alternating current (a.c.) electrical field. The phenomenon of cell process formation under the action of this force was investigated. Inhibitors of actin polymerization did not prevent generation of processes in the electrical field. At the early stages of cell spreading, the processes could be induced at any part of the cell membrane. After cell polarization was completed, protrusions could beformed at the active edge of the cell, but not at its stable edge. Pre-existing protrusions (but not retraction fibers) could be elongated by the external force. The results of these experiments demonstrate that different areas of the cell membrane differ in their ability to form processes under the action of a membrane-applied force. The significance of these data to the structure of the cortical layer is discussed.
Analysis of the distribution of the INCENPs throughout mitosis reveals the existence of a pathway of structural changes in the chromosomes during metaphase and early events in cleavage furrow formation
Authors: Earnshaw William C. , Cooke Carol A.
Published: April 2021

The INCENPs are two polypeptides of 135 ×103 and 150×103 Mr that enter mitosis as tightly bound chromosomal proteins, but subsequently leave the chromosomes altogether and become associated with the central spindle and cell cortex at the contractile ring. In the experiments reported here we have used confocal microscopy and immunoelectron microscopy to provide a detailed picture of the intracellular location of these proteins during mitosis. The experiments have not only revealed a number of new details concerning the properties of the INCENPs in mitosis, but have revealed a number of novel aspects of the mitotic process itself. The first of these is the existence of a sequential pathway of structural changes in the chromosomes that occurs during metaphase. This pathway is revealed by the existence of four distinct INCENP staining patterns in mitotic cells. In ‘early’ and ‘early/mid’ metaphase, the INCENPs gradually become concentrated at the centromeres, forming a ring at the center of the metaphase plate. During *mid/late’ metaphase they exit from the chromosomes, so that by late metaphase they are found solely in streaks that traverse the plate parallel to the spindle axis. The streaks probably correspond to INCENPs closely associated with microtubule bundles, perhaps as part of the stem body material. Examination of transverse optical sections of the spindle interzone during early anaphase reveals an unexpectedly high degree of order. The INCENP antigens are localized on fibers that are organized into a hollow ring 8 μm in diameter and ∼4μm beneath the cell cortex. Measurement of cellular dimensions in the confocal microscope reveals that the maximum diameter of early anaphase cells lies across the spindle equator, so that when the cleavage furrow forms, it does so around the maximum circumference of the cell. During anaphase, a subpopulation of the INCENP antigen becomes localized to the cortex where the furrow will subsequently form. This occurs prior to any other evidence of furrowing. Thus, binding of the INCENPs to this region may represent an early step in furrow formation. Together, these results suggest that the INCENPs may represent a new class of ‘chromosomal passenger’ proteins that are carried to the spindle equator by the chromosomes and subsequently perform a cytoskeletal role following their release from the chromosomes at the metaphase:anaphase transition.