bluuebunny/crossref_metadata_2025 huggingface dataset:
Changes in striated muscle fibres during contraction and growth with particular reference to myofibril splitting
Authors: Goldspink G.
Published: April 2021

Ultrastructural measurements were carried out on the mouse biceps brachii and soleus muscles fixed at different states of contraction and stretch. At a sarcomere length of 2·7–2·9 μm the more peripheral actin filaments ran slightly obliquely from the Z-disk to the A-band. This is due to a mismatch between the rhombic actin lattice at the Z-disk and the hexagonal lattice at the M-line. For a perfect transformation of a rhombic lattice into a hexagonal lattice the ratio of the lattice spacings has to be 1:1·51. However, at this sarcomere length the ratio is about 1:2·0 (Z:M). During contraction the angle of the peripheral actin filaments remains approxi-mately the same because the expansion of the M lattice is compensated for, partly by an increase in the Z-lattice spacing and partly by the bowing of the peripheral myosin filaments. When the sarcomeres are stretched beyond 3·0 μm the myosin filaments straighten out and the Z:M ratio decreases. The ratio of 1:1·51 is almost attained when there is no overlap of the actin and myosin filaments.

Ultrastructural measurements were also carried out on biceps brachii muscles of different ages. The lattice spacings for a standard sarcomere length did not change during the post-natal growth period. The amount of myofibrillar material and sarcoplasmic reticulum plus transverse tubular system were estimated using linear analysis for muscles at 3 different stages of growth. It was found that the myofibrillar cross-sectional area in an individual muscle fibre may increase 40-fold during growth and that the transverse tubular and sarcoplasmic reticulum systems increase at about the same rate.

In both the biceps brachii and the soleus muscles the myosin and actin filaments are not built into a continuous mass but they are divided into numerous discrete myofibrils. Subdivision of the myofibril mass occurs because the myofibrils split once they attain a certain size. The evidence presented in this paper supports the suggestion that the longitudinal splitting of the myofibrils occurs by the ripping of the Z-disks. When tension is rapidly developed by 2 adjacent sarco-meres a stress is produced at the centre of the Z-disk resulting from the oblique pull of the actin filaments. This causes some of the Z-disk filaments to rip and the rip then extends across the disk with the direction of the weave of the lattice. Evidence for the mechanism includes electron-micrographs showing Z-disks that are apparently just commencing to split; in these cases a hole can be seen in the centre of the disk. A model experiment is described which demonstrates the importance of the rate of tension development in causing myofibril splitting. Rapid tension development produces a snatch effect which causes the Z-disk filaments to break more readily. This may explain why the myofibrils in fast muscles tend to be small and discrete whilst those in slow muscles are larger and more irregular in shape.
Pollen-wall proteins: localization, enzymic and antigenic activity during development in Gladiolus (iridaceae)
Authors: Knox R.B.
Published: April 2021

Cytochemical methods have been used to follow the incorporation of enzymes and antigens in the cellulosic intine of the pollen grain of Gladiolus gandavensis. Acid phosphatase, esterase and ribonuclease were first detected in the developing intine during the vacuolate period, and by the end of this phase activity was present throughout the intine, especially in the area of the colpus, where this wall layer is thickened. Enzyme activity was also noted in the peripheral region of the protoplast during the period of intine growth. Succinic and NADH (reduced nicotine adeninedinucleotide) dehydrogenase activity was detected in the outer zone of the protoplast, and, at the end of pollen development, in the interbacular cavities of the exine and the pollen-coat materials, but at no time were these enzymes found in the intine.

The appearance of the antigens was traced during pollen development. Antigens were first detected in the early vacuolate period, soon after the release of the spores from the meiotic tetrads. They were found to be associated with the intine from the first deposition of poly-saccharides in this layer. As the intine thickened, so the amounts of associated antigens were found to increase, especially in the neighbourhood of the colpus.

In slurries prepared using mature pollen, enzymes and antigens were found to diffuse into the medium even with preparation times as short as 30 s. The leakage was over the whole of the exine, not only in the vicinity of the colpus; this may be related to the thinness of the exine in Gladiolus and the presence of pores in the tectum.

Electron micrographs showed the presence of vesiculate and tubular cavities in the intine, containing fibrillar or granular material. These inclusions are presumed to be the sites of the enzymes and antigens, since in a related genus, Crocus, acid phosphatase has been shown to be present in the corresponding cavities.

Gel diffusion and immunoelectrophoresis showed the presence of 4 major antigenic fractions in the leachates from mature Gladiolus pollen. Tests with the antisemm against leachates from species of the related genus, Iris, showed 1 cross-reacting fraction.
Zoospore germination in Blastocladiella emersonii: III. structural changes in relation to protein and rna synthesis
Authors: Soll D. R. , Sonneborn D. R.
Published: April 2021

Zoospores germinate rapidly and semi-synchronously upon exposure to growth medium or an inorganic salts solution. Amino acid incorporation into protein is detected only after a characteristic lag period, the extent of which is a function of developmental, rather than absolute, time. The ‘tum-on ‘of amino acid incorporation occurs after several of the morphological events of germination have taken place -notably, retraction of the flagellum, conversion to a spheroid cell morphology, vesicle fusion with the plasma membrane, formation of the initial cell wall, and elongation of the single mitochondrion. A second group of morphological changes -release of ribosomes from the nuclear cap into the cytoplasm, appearance of multiple mitochondrial profiles, disappearance of the flagellar axoneme, and disappearance of gamma particles -takes place in the cell population during the tum-on of incorporation. Cycloheximide reversibly inhibits germination at a characteristic block point. Inhibited cells accomplish all the known morphological events of germination except the disappearance of the flagellar axoneme and the formation of the germ tube. Amino acid incorporation is inhibited to undetectable levels in growth medium and by about 80% in the inorganic salts medium. Nevertheless, the population kinetics for the morphological changes which occur in cycloheximide-inhibited cells are indistinguishable from those in untreated cells. Cycloheximide effectively enters cells prior to the characteristic block point, since the drug drastically inhibits the low level of amino acid incorporation by zoospores. ’Pools’ extractable by trichloroacetic acid (TCA) have been examined; the results do not support the possibility of extensive early protein synthesis from endogenous sources. Actinomycin D, at levels which inhibit [3H]uridine incorporation into TCA-precipitable material (nucleic acid) by over 98 %, does not affect the population kinetics of germination. The drug does not affect the lag period for amino acid incorporation. The rate of amino acid incorporation (pulse labelling) fluctuates predictably during germination. Actinomycin D only partially inhibits the first ‘round ‘of incorporation, but severely inhibits the second.

From this information, we conclude that: (a) the proteins necessary for most of the structural events of germination are preformed in the zoospore; (f>) therefore, these events are controlled directly by mechanisms other than differential protein synthesis; (c) two of the known events -disappearance of the flagellar axoneme and formation of the germ tube -do appear to have a requirement for concomitant protein synthesis; (d) germination does not require de novo RNA synthesis; and (e) certain of the first proteins made during germination are evidently synthesized with preformed messages.
The in vivo and ultrastructural effects of injection of lysophosphatidyl choline into myelinated peripheral nerve fibres of the adult mouse
Authors: Hall Susan M. , Gregson N. A.
Published: April 2021

The action of phospholipase A and lysophosphatidyl choline (LPC) on mature, myelinated peripheral nerve fibres has been studied in vivo and electron microscopically, following sub- perineurial injection of these substances. Within 30 min, demyelination was observed in vivo along cylindrico-conical segments, spreading from Schmidt-Lanterman incisures and nodes of Ranvier. By 96 h, all traces of the myelin sheath had disappeared from the area of the lesion, and had been replaced by debris-laden cells lying in chains parallel to one another and the long axis of the fibre. During the next few weeks these cells gradually disappeared, and numerous finely myelinated axons, running between, and in continuity with, the normal fibres proximal and distal to the lesion were observed. If lower concentrations of LPC were used the number of fibres involved decreased, although the demyelinative changes followed the same time-course.

Ultrastructurally, demyelination involved progressive disruption and removal of the lamellar sheath, observed initially as a splitting of the intraperiod line within 30 min. Subsequent breakdown resulted in the formation of strands of 4-6 nm repeat material which was further degraded through quintuple- and triple-layered lamellar units to foam-like systems of disorganized lamellar fragments. The Schwann cell and axons appeared to be undamaged by phospholipase A and LPC, and retained their normal impermeability to exogenous ferritin.

The significance of the demyelinating capacity of LPC in vivo is discussed in terms of its known action on myelin in vitro, the rapidity and apparent specificity of its action demonstrated in this study, and its potential involvement in pathological demyelination.
Pachytene spermatocyte and round spermatid binding to sertoli cells in vitro
Authors: Enders George C. , Millette Clarke F.
Published: April 2021

Spermatogenic cells differentiate in vivo while in continuous contact with the Sertoli cell. During differentiation, the spermatogenic cells and Sertoli cells form a number of morphologically distinct stage-specific adhesions. We describe an in vitro assay system for studying the adhesion of spermatogenic cells to Sertoli cell monolayers. Mixed populations of spermatogenic cells or enriched fractions of pachytene spermatocytes and round spermatids were labelled with the vital dye, fluorescein diacetate, prior to their addition to Sertoli cell monolayers so that the adhesion of viable spermatogenic cells could be quantified. Using this assay system, the number of pachytene spermatocyte and round spermatid binding sites on the Sertoli cell monolayer were similar, but the kinetics of binding were different. Pachytene spermatocytes were able to inhibit significantly round spermatid binding, while round spermatids did not significantly inhibit pachytene spermatocyte binding. After coculture for 24–48 h, spermatocytes form junctional structures with Sertoli cells that are similar to desmosome-like junctions. These results suggest that pachytene spermatocytes and round spermatids bind to Sertoli cells by different mechanisms.
The somatic function of the micronucleus in sexual reproduction of Paramecium tetraurelia: Initiation of oral membranelie assembly
Authors: Chau M. F. , Ng Stephen F.
Published: April 2021

In a previous study, cell lines possessing defective micronuclei, generated by laser-microbeam irradiation, gave rise to cells lacking both oral apparatus and micronuclear derivatives after autogamy. It was concluded that astomy arose as a result of degeneration of all of the meiotic products of the micronuclei after meiotic telophase II, instead of leaving one product for subsequent nuclear reorganization. The present study consolidates this conclusion by employing 15 micronucleus-defective cell lines; these were generated by laser-irradiation of the micronucleus, treatment of the cells with cis-dichloro-diammineplatinum (II), and conjugation between diploids and amicronucleates to produce haploids. A good correlation between the presence of pregametic, gametic and zygotic nuclei and the initiation of oral membranelie assembly in stomatogenesis was demonstrated in 17 cases of autogamy. Therefore, postmeiotic micronuclear activities up to the zygotic nucleus stage, in particular in the gametic stage, are crucial for the initiation of oral membranelie assembly, while premeiotic micronuclear activities are insufficient. Micronuclear genic factors are also likely to be involved in the determination of the fate of the meiotic products.
A morphologically distinct candidate for an epithelial stem cell in mouse mammary gland
Authors: Smith Gilbert H. , Medina Daniel
Published: April 2021

Transplantation studies demonstrate that an epithelial stem cell component must exist in the mouse mammary gland throughout life. Samples taken from any portion of the mammary gland at any age and at any developmental stage, including full functional differentiation, give rise to mammary epithelial outgrowths with complete developmental capacity. Cytological examination of mouse mammary gland explants revealed the presence of morphologically distinct cells distributed sporadically among the mammary epithelium, whose behaviour in vivo and in vitro suggested that they might represent a latent epithelial stem cell population. These pale-staining cells possessed large spherical nuclei, a clear cytoplasm and a round smooth-contoured shape. Electron microscopy confirmed their pale-staining characteristics and revealed a cytoplasm sparsely populated with organellar structures, such as mitochondria and endoplasmic reticulum. Their epithelial genealogy was demonstrated by the presence of terminal bars and tight junctions formed with their epithelial cell neighbours. In vivo, these cells were found among mammary epithelial cell populations in 16-day-old embryos onward in both ductal or lobular structures during all stages of pregnancy, lactation and involution. In explant cultures, these cells did not undertake a secretory morphology in the presence of lactogenic hormones, although occasionally they became immunologically positive for casein. They did not incorporate [3H]-thymidine into their nuclei under any of the experimental conditions used; however, they appeared to undergo mitosis within 4 h regardless of the presence or absence of hormone(s). At 24 h increased numbers of pale cells were found in pairs or in groups. At 72 h in the presence of IFPrl (medium containing insulin, hydrocortisone and prolactin), the pairs and groups of pale cells observed at 24 h were not found. Instead, individual pale cells were seen among groups of cytologically and functionally differentiated secretory epithelial cells. When lactogenic hormones were not present, groups of pale cells were still present in the explants at 72 h. These findings suggest that the pale cells are arrested at Gz phase of the cell cycle and that they give rise by mitosis to daughter cells capable of differentiating in the presence of lactogenic stimuli. Inhibition of DNA synthesis in the explants did not alter these cellular events.
A Dictyostelium mutant with severe defects in α-actinin: its characterization using cDNA probes and monoclonal antibodies
Authors: Schleicher Michael , Noegel Angelika , Schwarz Tobias , Raff Eva Wall , Brink Marijke , Faix Jan , Geriscii Glinther , Isenberg Gerhard
Published: April 2021

Cells of a Dictyostelium discoideum mutant de­ficient in binding a monoclonal antibody to α·actinin have previously been shown to grow and develop similarly to the wild type and to exert unimpaired chemotaxis as well as patching and capping of membrane proteins. Here we show that the normal 3.0 kb message for α-actinin is replaced in the mutant by two RNA species of approximately 3.1 and 2.8 kb. The 3-1 kb RNA was recognized by DNA fragments from all parts of the coding region, while the 2.8 kb RNA hybrid­ized to all but a 3'-terminal fragment. Proteins synthesized in the mutant were analysed using four monoclonal antibodies that in the wild type specifically recognize the 95×l03Mr polypeptide of α-actinin. Cleavage mapping indicated that the binding sites of these antibodies are distributed over a region comprising more than half of the α- actinin polypeptide chain. In the mutant, three of the antibodies faintly labelled two polypeptides of 95×l03Mr and 88×l03Mr; the fourth antibody, which binds closest to one end of the polypeptide chain, faintly labelled the 95×l03Mr polypeptide only. The 88×l03Mr polypeptide most probably lacks the C-terminal portion of α-actinin.

The binding of an antibody that recognized both polypeptides was quantified by a radio- immuno competition assay using wild-type α- actinin as a reference. In a mutant cell extract containing total soluble proteins the antibody binding activity was decreased to 1.1% when compared with wild-type extract. After their par­tial purification and SDS–polyacrylamide gel electrophoresis the mutant 95 × 103Mr and 88 × 103Mr polypeptides were barely detectable as Coomassie Blue-stained bands, indicating that in the mutant not only certain epitopes of α-actinin were altered but the entire molecule is almost completely lacking. When the fitness of mutant cells relative to wild type was determined during growth in nutrient medium, a slight disadvantage for the mutant was indicated, by finding selection coefficients between 0.03 and 0.05.
Cultured epithelial cells derived from human foetal pancreas as a model for the study of cystic fibrosis: Further analyses on the origins and nature of the cell types
Authors: Harris Ann , Coleman Lindsay
Published: April 2021

The establishment of a tissue-culture system for epithelial cells derived from human foetal pancreas has recently been reported. Further analyses have now been made on these cells in vitro, together with parallel investigation of the distribution of different cell types within the intact foetal pancreas. Results support the view that the cultured cells are ductal in origin and nature. Pancreatic epithelial cell cultures have also been established from foetuses with cystic fibrosis.
Differences in cytoplasmic ph and the sensitivity to acid load between prespore cells and prestalk cells of Dictyostelium
Authors: Inouye Kei
Published: April 2021

The relationship between cytoplasmic pH (pH)) and prestalk/prespore differentiation in the cellular slime mould Dictyostelium discoideum was investigated by: (1) comparing the pHi of prestalk and prespore cells, and (2) examining the effects on pH; of weak acids, weak bases and proton-pump inhibitors. These substances are known to affect the spore-to-stalk ratio. The pHi of dissociated slug cells was measured by microfluorometry using a pH-sensitive fluorescence dye, carboxyfluorescein.

There was a slight but significant difference in pHi between prestalk cells (7’38 ± 0·09) and prespore cells (7·49 ± 0·03), and prestalk cells were more sensitive to acid load than prespore cells. In addition, weak acids and proton-pump inhibitors, which promote prestalk differentiation, significantly lowered pHi whereas weak bases, which induce prespore differentiation, elevated pHi. However, differentiation inducing factor (DIF), an endogenous inducer of stalk differentiation, caused only a slight and transient decrease in pHi. These results suggest two alternative possibilities: (1) a small and transient decrease in pHi is sufficient to induce prestalk differentiation, or (2) there is a mediator other than pHi that links the action of DIF and the pathway choice.
Antibodies to rat sertoli cell secretory proteins recognize antigenic sites in acrosome and tail of developing spermatids and sperm
Authors: Kierszenbaum Abraham L. , Ueda Hiroshi , Ping Liu , Abdullah Munir , Tres Laura L.
Published: April 2021

Polyclonal antisera raised against polypeptide components of antigenically related rat Sertoli cell secretory protein S70 and S45–S35 heterodimeric protein were used to identify antigenic sites in rat testis during spermatogenesis. Immunofluorescence, immunoperoxidase and immunogold electron-microscopic methods have shown that all antisera recognize immunoreactive sites in the acrosome of developing spermatids. Apical cytoplasmic regions of Sertoli cells in close association with bundles of developing spermatids displayed an immunoreactive product. The principal piece of the developing tail of maturing spermatids immunostained with antisera to Sertoli cell secretory S45–S35 heterodimeric protein, displayed characteristic apical-to-distal immunoreactive gradient patterns before acquiring uniform immunoreactivity at completion of maturation. Immunogold electron microscopy demonstrated that outer dense fibres were the predominant immunoreactive site. Results of this work, together with previous immunoblotting and immunofluorescent data, demonstrate that both Sertoli cell secretory proteins and components of the acrosome and tail of developing spermatids and epididymal sperm share antigenic homology.
In vitro modulation of endothelial fenestrae: Opposing effects of retinoic acid and transforming grow h factor β
Authors: Lombardi T. , Montesano R. , Furie M. B. , Silverstein S. C. , Orci L.
Published: April 2021

Cultured endothelial cells isolated from fenestrated capillaries express many properties characteristic of their in vivo differentiated phenotype, including the formation of a limited number of fenestrae. In this study, we have investigated whether physiological factors that control cell differentiation might regulate the surface density of fenestrae in capillary endothelial cells. We have found that treatment of the cultures with retinoic acid (10μM) induces a more than threefold increase in the surface density of endothelial fenestrae, whereastransforming growth factor β (TGFβ) (2 ng ml−1) causes a sevenfold decrease in the surface density of these structures. These results show that the expression of endothelial fenestrae is susceptible to bidirectional modulation by physiological signals, and suggest that retinoids and TGFβ may participate in the regulation of fenestral density of capillary endothelium in vivo.
Transferrin receptors on the surfaces of retinal pigment epithelial cells are associated with the cytoskeleton
Authors: Hunt Richard C. , Dewey Art , Davis Alberta A.
Published: April 2021

Retinal pigment epithelial cells, derived from human donor eyes, have been grown in culture as monolayers on membrane filters or plastic surfaces and shown to possess transferrin receptors with a monomeric molecular mass of 93000. These receptors internalize 12SI-labelled transferrin and recycle it to the surrounding medium in a similar manner to other cell types. Scatchard analyses show that there are about 100 000 high-affinity receptors on the surface of each cell and most of these receptors are associated with the cytoskeleton. In total cell extracts, there are additional low-affinity binding sites that do not appear to be strongly associated with the cytoskeleton. The apparent interaction of transferrin receptors with the cytoskeleton was confirmed in two ways: first, using 200 kV electron microscopy for stereo analyses, skeleton-associated transferrin receptors were detected by a monoclonal anti-receptor antibody and a colloidal gold-conjugated second antibody after Triton X-100 extraction of pigment epithelial cells grown directly on laminin-coated gold grids; and, second, when cell surface receptors were labelled with radioiodinated transferrin and then incubated for various periods of time, the labelled transferrin was observed to move from a Triton X-100-insoluble fraction (a putative cytoskeletal compartment) to a Triton-soluble compartment that was not associated with the cytoskeleton. Using either horseradish peroxidase or colloidal gold-labelled transferrin, it has been shown that basolateral and apical surface-located receptors participate in receptor-mediated endocytosis via clathrin-coated pits, endosomes and tubular structures. Initially, transferrin internalized from the apical surface is observed in small endosomes that often appear to be embedded in an apical layer of microfilaments. From these peripheral regions of the cells, the labelled receptors move to larger endosomes and multivesicular bodies deeper in the cytoplasm. These structures have no apparent association with cytoskeletal elements.
Tandem scanning reflected-light microscopy of cell-substratum adhesions and stress fibres in swiss 3T3 cells
Authors: Paddock Stephen W.
Published: April 2021

This paper describes two applications of the tandem scanning reflected-light microscope (TSM) for the observation of the structure of individual cells growing in tissue culture. First, the TSM is used as an alternative to interference reflection microscopy (IRM) or total internal reflection aqueous fluorescence microscopy (TIRAF) to observe cell-substratum adhesions in unstained living cells growing on a glass coverslip. Second, the TSM is used to produce improved images of cellular structures in 3T3 cells stained with various protein dyes including Napthol Blue Black (NBB) and Coomassie Brilliant Blue (CBB). More specifically, close contacts and focal contacts are resolved in living 3T3 cells, and features of the nucleus, the cytoskeleton and extracellular matrix are resolved in both NBB- and CBB-stained cells. The focal contacts and associated stress fibres are clearly imaged in NBB-stained cells. The TSM is an improvement over conventional incident light microscopy because the confocal image excludes information from out-of-focus regions of the cytoplasm, and, unlike the laserbased confocal microscope, the actual colour of the specimen is viewed directly with the TSM in almost real-time.
Cross-linking of microtubules by microtubule-associated proteins (MAPs) from the brine shrimp, Artemia
Authors: Campbell Elizabeth J. , Mackinlay Sara Ann , Macrae Thomas H.
Published: April 2021

Microtubules induced with taxol to assemble in cell-free extracts of the brine shrimp, Artemia, are cross-linked by microtubule-associated proteins (MAPs). When the MAPs, extracted from taxol-stabilized microtubules with lM-NaCl are co-assembled with purified Artemia or mammalian neural tubulin, reconstitution of cross-linking between microtubules occurs. The most prominent non-tubulin protein associated with reconstituted cross-linked microtubules has a molecular weight of 49000 but we cannot yet exclude the possibility that other proteins may be responsible for the crosslinking. Cross-linkers are separated by varying distances while cross-linked microtubules, pre pared under different conditions, are 6·9-7·7nm apart. Cross-linking of microtubules by MAPs occurs whether MAPs are added to assembling tubulin or to microtubules, and it is not disrupted by ATP. The MAPs are heat-sensitive and do not stabilize microtubules to cold. Immunological characterization of Artemia MAPs on Western blots indicates that Artemia lack MAP 1, MAP 2 and tau. Our results clearly demonstrate that Artemia contain novel MAPs with the ability to cross-link microtubules from phylogenetically disparate organisms in an ATP-independent manner.
Synthesis and phosphorylation of cytoskeletal proteins during the in vitro biogenesis of MDCK cell monolayers
Authors: Benítez-King G. , Cazares F. , Meza I.
Published: April 2021

In vitro formation of a functional MDCK cell monolayer requires the dynamic participation of the cytoskeleton. Cell shape, contacts and polarity, as well as transepithelial electric resistance (TER), are actively modified during this differentiation process. We studied the distribution and rearrangement of cytokeratin, vimentin and actin filaments that occur in the monolayer concomitant with the synthesis and phosphorylation of these proteins. Cells cultured for short time in suspension, subconfluent or early confluent cells, showed striking differences in cell shape and arrangement of their cytoskeletal filaments. Subconfluent and early confluent cells synthesized proteins at high levels. In contrast, suspended cells maintained lower rates of protein synthesis. Labeled amino acid uptake was very similar in all these culture conditions. Gel electrophoresis analyses of the synthesized proteins showed increases in the synthesis of actin, vimentin and specific cytokeratins in subconfluent and early confluent cells. On the other hand a decrease in total protein, actin, vimentin and cytokeratin synthesis was observed in cells kept in suspension for 24 h or in 78-h or 174-h older confluent cultures. These cultures also showed a decrease in the uptake of labeled amino acid. Cytokeratin and vimentin phosphorylation rates were also modified during the in vitro formation of a monolayer. In suspended cells, cytokeratins were phosphorylated and three labeled isoelectric variants of the 40, 48 and 58K (K = 103Mr) cytokeratins were present in intermediate filament extracts. In subconfluent and early confluent cells only two isoelectric phosphorylated variants of the 40, 48 and 58K cytokeratins were detected and vimentin was also phosphorylated. In confluent monolayers cultured 78 and 174 h, only 40 and 48K cytokeratins remained labeled, while the 58K cytokeratin and vimentin, although present in the cell extracts, were no longer phosphorylated. Our results indicate that concerted changes occur in the distribution of intermediate and actin filaments, and the relative rate of synthesis and phosphorylation of cytoskeletal proteins during formation of an MDCK cell functional monolayer.
Reduction of G1 phase duration and enhancement of c-myc gene expression in HeLa cells at hypergravity
Authors: Kumei Yasuhiro , Nakajima Tohru , Sato Atsushige , Kamata Nobuyuki , Enomoto Shoji
Published: April 2021

We have found that hypergravity stimulates the proliferation of HeLa cells through reduction of the Gt phase duration, concomitant with enhancement of c-myc gene expression. HeLa cells were grown in monolayer in culture flasks that were centrifuged to generate a constant 18, 35 or 70g at 37°C for up to 4 days. The cell proliferation was enhanced at 18, 35 and 70g, most notably at 35 g. Cell cycle analyses with [3H] thymidine (TdR)-colcemid treatment showed that the cell generation time in the 35 g culture was reduced by 17% as compared to the control, which was attributed to a 26% reduction of the Gi phase duration. No differences were observed in the duration of the S, G2 and M phases or in the [3H]TdR incorporation per S phase cell between the 35g culture and the control. The induction of c-myc gene expression was investigated by

RNA blot hybridization during a 15–360 min exposure of cells to 18, 35 and 70g. Elevated levels of c-myc mRNA were observed after a 15-min exposure, and maintained after a 360-min exposure at all hypergravities examined. The highest induction rate of c-myc mRNA was 3·8-fold higher than the control after a 120-min exposure to 35 g. The 35g condition was the most effective hypergravity for stimulating both cell proliferation and c-myc gene expression. Our study suggests that the appropriate level of hypergravity stimulates HeLa cell proliferation by reducing the G1 phase duration without affecting DNA synthesis rate, mediated through induction of c-myc gene expression.
A simple formulation of microtubule dynamics: quantitative implications of the dynamic instability of microtubule populations in vivo and in vitro
Authors: Bayley Peter M. , Schilstra Maria J. , Martin Stephen R.
Published: April 2021

A simple formulation of microtubule dynamic instability is presented, which is based on the experimental observations by T. Horio and H. Hotani of coexisting, interconverting growing and shrinking microtubules. Employing only three independent, experimentally determined parameters for a given microtubule end, this treatment accounts quantitatively for the principal features of the observed dynamic behaviour of steady-state tubulin microtubules in vitro. Experimental data are readily reproduced for microtubule length redistribution, and for the kinetics of tubulin exchange processes, including pulse-chase properties. The relative importance of dynamic incorporation and that due to treadmilling are assessed. Dynamic incorporation is found to dominate the overall exchange properties; polarized incorporation due to treadmilling generally becomes significant only when the dynamics are largely suppressed.

This treatment also permits simulation of certain cellular phenomena, showing how microtubule renucleation can control microtubule growth, by means of changes in microtubule number concentration in a system at constant microtubule mass.

A relatively simple extension of the formulation accounts quantitatively for non-steady-state microtubule properties, e.g. dilution-induced rapid disassembly and the oscillatory mode of microtubule assembly. The principles relating dynamic instability and oscillatory behaviour are clearly indicated. Possible mechanisms of the switching of microtubules are briefly discussed.
Differences in intracellular transport of a fluorescent phosphatidylcholine analog in established cell lines
Authors: Sleight Richard G. , Abanto Marina N.
Published: April 2021

The transport and metabolism of a fluorescent phosphatidylcholine analog, l-palmitoyl-2-(N-4-nitrobenzo-2-oxa-l,3-diazole)-aminocaproyl-phos-phatidylcholine ((palmitoyl, C6-NBD)-PC), in BHK, CH0-K1, CHO-15B, MDCK, VA-2, Vero, V79 and WI-38 cells has been investigated. When liposomes containing (palmitoyl, C6-NBD)-PC were incubated with cells at 2°C, spontaneous transfer of the fluorescent lipid from the liposomes to the cells’ plasma membranes occurred. Most of the lipid transferred to the cells could be removed by incubating the cells in the presence of nonfluorescent liposomes or media containing 10% serum, suggesting that the fluorescent probe resided exclusively in the outer leaflet of the plasma membrane at 2°C.

After insertion into the plasma membrane, internalization of (palmitoyl, Ce-NBD)-PC occurred when the cells were warmed to 37°C. This resulted in four different labeling patterns: (1) little or no internalization of (palmitoyl, C6-NBD)-PC into punctate vesicles was observed in Vero cells. (2) Transport of (palmitoyl, C6-NBD)-PC to the region of the Golgi apparatus and to a small number of intracellular vesicles was observed in both V79 and CHO-K1 cell lines. (3) A large number of fluorescently labeled intracellular vesicles with little or no labeling in the region of the Golgi apparatus appeared after the internalization of (palmitoyl, C6-NBD)-PC in BHK, CHO-15B, MDCK and WI-38 cell lines. (4) Accumulation of (palmitoyl, C6-NBD)-PC in small vesicles, mitochondria and the nuclear envelope was observed in VA-2 cells. In addition, cells having a defect in glycoprotein processing and those transformed with simian virus 40 (SV40) internalized the fluorescent lipid probe differently compared with parental lines. Neither differences in rates of endocytosis nor rates of (palmitoyl, C6-NBD)-PC degradation between cell types appears to cause the observed dissimilarities in intracellular lipid transport. We suggest that these different cell types may have dissimilar pathways of intracellular lipid trafficking or differential regulation of a common transport pathway, and that the predominant pathway of lipid translocation can be altered in cells by changing the composition of their glycoproteins or by viral transformation.
Changes in nuclear distribution of large tumor antigen correlate with the proliferative activity of simian virus 40-transformed cells
Authors: Ordenes Gamaliel E. , Beck Ingrid , Carmona Maria T. , Santos Myriam
Published: April 2021

A correlation between intranuclear distribution of large tumor antigen (T-ag) and proliferative activity in Simian virus 40 (SV40)-transformed cells has been established. Nuclear T-ag was cytochemically detected by the immunoperoxidase reaction. Cell populations with a high growth rate displayed a heterogeneous pattern with patches of positive reaction surrounded by large and irregular negative areas. By contrast, cell populations with a low growth rate presented a rather homogeneous distribution of nuclear T-ag with a finely granular positive reaction all over the nucleus, excluding nucleolus-like areas. The latter pattern was also observed in cells that had been inhibited from traversing S phase. In this case, the heterogeneous distribution of T-ag within the nucleus was recovered once the cells were allowed to proceed again through S phase, or through G2 phase. Highly condensed chromatin in mitotic chromosomes appeared to exclude T-ag fully as early as in prophase. Then, nuclear distribution of T-ag changes throughout the cell cycle, thus suggesting it might be a cell cycle-dependent event. Interestingly, thymidine-labeled cells from cultures with a high growth rate presented a heterogeneous distribution of autoradiographic grains. Regardless of their mechanism(s) of origin, T-ag-enriched areas might correspond to nuclear sites where a specific function, such as DNA synthesis, is being carried out.
5-Azacytidine-induced alterations in the GH12C1 cells: effects on cellular morphology, chromosome structure, DNA and protein synthesis
Authors: Parrow Vendela C. , Aleström Peter , Gautvik Kaare M.
Published: April 2021

Growth hormone-producing rat pituitary tumor cells (GH12C1) were cultivated in the presence of 5-azacytidine (5-AC). After cessation of treatment the cells were allowed to recover in normal cell culture medium prior to subcultivation. Within hours after the subcultivation the cells underwent several morphological alterations, later followed by changes in growth pattern: (1) cell shape was irreversibly changed from round or spindle shape to different clones of fibroblast-like cells; (2) some of these clones later formed foci; and (3) an extensive generation of multinucleated cells was seen. The demethylation was maximal approximately one week after the first subcultivation in the absence of 5-AC.

Severe alterations of the chromosome structure were seen after the first subcultivation. During the following weeks the 5-AC-treated cells showed impaired chromosome condensation, and homogeneous staining with quinacrine mustard. During this period the DNA synthesis is doubled when compared to control cell DNA synthesis. The core histone synthesis increases in parallel with the DNA synthesis, but the majority of nuclear proteins, including the linker histones, remain at the control level. This results in a ratio of linker histone to core histone synthesis at approximately half the control cell level. The altered ratio of synthesis slowly decreased to control levels during a period of five weeks of continuous cultivation in the absence of the drug, and the under-condensed chromosomes could no longer be seen. The induced novel phenotypes with their pleiomorphic appearances were conserved.

The growth hormone synthesis remained constant during all phases of the experiment and prolactin synthesis was not induced.
Inositol tris- and polyphosphate formation during chemotaxis of Dictyostelium
Authors: Europe-Finner G. N. , Gammon B. , Wood C. A. , Newell P. C.
Published: April 2021

Using the technique of HPLC with Partisil SAX columns, we have found that stimulation of amoebae of Dictyostelium discoideum with the chemoattractant cyclic AMP induces the rapid accumulation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), with a peak at 5s. A smaller HPLC peak (designated P3) that elutes just after the Ins(1,4,5)P3 peak accumulates more slowly to a maximum at 20 s. In control studies, the changes in Ins(1,4,5)P3 were shown not to be due to varying recovery from the cell extracts and a comparison of reverse-phase and Partisil SAX HPLC columns showed similar values for determinations by either method.

The involvement of a G-protein in this chemotactic system was confirmed by the finding that accumulation of Ins(1,4,5)P3 was elicited by the addition of GTP γS (5′-[gamma-thio]triphosphate) to saponin-permeabilized amoebae.

A study of the changes in the lipid-soluble phosphatidyl inositol phosphates demonstrated that cyclic AMP also stimulated a rapid loss of radioactivity from 32P-labelled phosphatidylinositol 4,5-bisphos-phate (PtdIns(4,5)P2), which corresponded in its timing to the rise in Ins(1,4,5)P3, indicating that a phosphoinositidase C (phospholipase C) is present that can be stimulated by occupation of the cell surface cyclic AMP receptors.
Studies on the mechanism of embryonic frog nerve orientation in a small applied electric field
Authors: Mccaig Colin D.
Published: April 2021

The mechanism of nerve orientation in an applied electric field has been investigated using a number of pharmacological agents. Galvanotropism may depend on redistribution within the plasma membrane of integral membrane proteins (IMP); blocking this with concanavalin A inhibited orientation. Orientation may depend also on an influx of Ca2+; Co2+ and La3+ blockade of calcium channels inhibited turning in an electric field. Organic blockers of calcium channels did not influence orientation, suggesting that L-type Ca2+ channels may not be present at the growth cone. Procedures that may induce asymmetric entry of Ca2+ on the anodal side of cells caused a reversal of normal galvanotropism, with growth directed towards the anode. This may implicate local levels of cytoplas mic Ca2+ within the growth cone in controlling turning behaviour. An asymmetric distribution of filopodia precedes and may predict the direction of nerve growth in an electric field. Various pharmacological agents perturbed the distribution of filopodia in such a way that this did not reflect subsequent orientation. It is suggested that, normally, local Ca2+ increases and an asymmetry of filopodia operate together in determining orientation, but that filopodial activity is subordinate to and can be overriden by local Ca2+ levels in the growth cone. In addition, two of the drug treatments markedly increased rates of nerve growth, which may be of importance in nerve regeneration.
Anaphase onset and dephosphorylation of mitotic phosphoproteins occur concomitantly
Authors: Vandré Dale D. , Borisy Gary G.
Published: April 2021

The cyclical phosphorylation and dephosphorylation of the centrosome during mitosis was analyzed by immunofluorescence methods using the MPM-2 antibody, which reacts with a subset of mitotic phosphoproteins. Quantification of MPM-reactivity indicated that centrosomal phosphorylation attained a maximal level just prior to ana-phase onset. This level was maintained in meta-phase cells blocked from further mitotic progression with the microtubule depolymerizing agent nocodazole. However, when nocodazole was added to cells that had just initiated anaphase, the level of centrosomal phosphorylation decreased rapidly as in untreated anaphase cells. We conclude that the onset of dephosphorylation of the centrosome coincided with the onset of anaphase and continued in the absence of chromosome movement. Dephosphorylation of MPM-2 reactive phos-phoproteins may be taken as a biochemical indicator of anaphase onset.
Circadian changes in cyclic amp levels in synchronously dividing and stationary-phase cultures of the achlorophyllous ZC mutant of Euglena gracilis
Authors: Carré Isabelle A. , Laval-martin Danielle l. , Edmunds Leland n.
Published: April 2021

Oscillations in adenosine 3′,5′-cyclic monophos-phate (cyclic AMP) level have been proposed to be part of the biochemical feed-back 100p(s), or *clock(s)’, believed to underlie circadian rhythmicity. This possibility has been examined for a cellular circadian oscillator in synchronously dividing (or nondividing) cultures of the photosynthesis-deficient ZC mutant of the alga Euglena gracilis Klebs (Z). We have demonstrated a bimo-dal, autonomously oscillating, circadian variation of cyclic AMP content in this unicell. Rhythmic changes of the cyclic AMP level, which may reflect the transition of the cell population through the different phases of the cell division cycle (CDC) in division-phased cultures, also persisted after the culture medium had become limiting and the cells had stopped dividing. We have also shown that the free-running, circadian oscillation of cyclic AMP content displayed by nondividing cells in continuous darkness could be phase-shifted by a light signal (a property inherent to most circadian systems), in a manner that could be predicted from the phase-response curve previously obtained for the cell division rhythm in the ZC mutant. These results suggest a possible role for cyclic AMP, either as an element of the coupling pathway for the control of the CDC by the circadian oscillator, or as a ‘gear’ of the clock itself.
Characterization of mitotic motors by their relative sensitivity to amp-pnp
Authors: Lee Greta Marlene
Published: April 2021

The relative sensitivities of the motors for mitotic chromosome movements and saltatory motion were compared using a nonhydrolyzable analog of ATP, AMP-PNP. K+AMP-PNP was microinjected into PtKl cells at the time of nuclear envelope disassembly or at anaphase onset. To produce a dose-response curve for the effect of AMP-PNP on the rate of movement, the intracellular concentration of AMP-PNP in individual cells was measured. The volume injected into each cell was determined by adding dextrans labeled with Lucifer Yellow to the injection buffer, measuring the injected cell’s fluor-escence intensity, and then comparing the value with the fluorescence intensity of known volumes of Lucifer Yellow dextran solution. AMP-PNP produced a 50% inhibition of spindle elongation at 0.2 mM, of saltatory motion at 0.8 mM, and of chromosome movement at 8.6 mM. Prometaphase chromosome movement and anaphase chromosome-to-pole movement were similarly inhibited by AMP-PNP. Equivalent volumes of injection buffer containing 1% Lucifer Yellow dextran had no effect on chromosome movement, spindle elongation or saltatory motion. Although AMP-PNP occasionally produced shorter anaphase spindles, tubulin immunofluorescence revealed the presence of abundant spindle microtubules. Metaphase cells treated with very high cell concentrations of AMP-PNP had spindles with unusually long astral microtubules; thus microtubules are stabilized rather than broken down by AMP-PNP. In conclusion, spindle elongation is four times more sensitive than saltatory motion to AMP-PNP and 40 times more sensitive than chromosome movement. When these sensitivities to AMP-PNP are considered with the results from other studies, it can be concluded that the molecular motors for spindle elongation, chromosome movement and saltatory motion are different.
Measurement of volume injected into individual cells by quantitative fluorescence microscopy
Authors: Lee Greta Marlene
Published: April 2021

Pressure microinjection is frequently used to introduce substances into mammalian cells, but precise quantitation of the volume injected into individual cells has been difficult. A simple and reliable procedure for determining the volume injected was developed in order to determine what intracellular concentration of AMP-PNP was necessary to inhibit specific cellular processes. The technique uses fluorescent Lucifer Yellow-labeled dextrans in the microinjection buffer and quantitative fluorescence microscopy to measure the fluorescence intensity of the injected cell. The volume injected is computed from a standard curve derived from the volume and fluorescence of spherical, microscopic droplets of Lucifer Yellow dextran solution. The droplets are ejected from a micropipet into immersion oil where they sink to rest on a siliconized coverslip. For the measurement of fluorescence, an inexpensive photomultiplier system that is attached to a fluorescence microscope is described. The potential uses of this method for other microassays are discussed.