bluuebunny/crossref_metadata_2025 huggingface dataset:
Fine structure study of pollen development in Haemanthus katherinae baker: III. changes in organelles during development of the vegetative cell
Authors: Sanger Jean M. , Jackson W. T.
Published: April 2021

The newly formed vegetative cell of the pollen grain of the African blood lily has a spheroidal nucleus, few dictyosomes, and a small amount of endoplasmic reticulum. Plastids are smaller than those of the microspore and usually lack starch granules. Mitochondria and lipid bodies are more numerous than they were in the microspores, but their appearance is unchanged. As the pollen grain matures, the vegetative nucleus becomes irregular in shape. There is a dramatic increase in the number of dictyosomes, starch accumulates in the plastids, and a moderately well developed system of endoplasmic reticulum appears in the form of fiat cisternae. In the developmental period immediately preceding anthesis, the vegetative nucleus becomes lobate and small nucleoli replace the large nucleoli present earlier. Plastids lose their starch, lipid bodies disappear, and the endoplasmic reticulum becomes vesicular in this final stage before germination.
The contractile mechanism in obliquely striated body wall muscle of the earthworm, Lumbricus terrestris
Authors: Knapp M. F. , Mill P. J.
Published: April 2021

Obliquely striated muscle fibres from the longitudinal and circular layers of the body wall of the earthworm were prepared in extended and contracted states for study in the electron microscope.

Contracted fibres differ from extended ones in the following respects: (i) the I-bands are narrower, (ii) the A-bands are wider, and (iii) there are more rows of thick myofilaments in each A-band.

The arrangement of the thick and thin myofilaments in interdigitating arrays and the occurrence of cross-links between the 2 types of myofilament indicate a classical sliding-filament mechanism of contraction as in cross-striated muscle, resulting in a reduction in the I-band width.

The increase in the A-band width could be due to a moving apart of the myofilaments during contraction to preserve constant volume of the lattice.

The third change, the increase in the number of rows of thick myofilaments in the A-band, can be explained only by a shearing of these filaments past one another in such a way as to increase the amount of their overlap.

The role of the sliding-filament and shearing contraction mechanisms in bringing about the changes observed in earthworm muscle fibres is considered and the possible correlation of these mechanisms with certain physiological data is discussed. The function of the sarcoplasmic reticulum in the transmission of impulses to the interior of the fibre and/or in the control of the contraction mechanism is also discussed.
Surface features of smooth muscle cells from the mesenteric artery and vas deferens
Authors: Devine C. E. , Simpson F. O. , Bertaud W. S.
Published: April 2021

Smooth muscle cells of small mesenteric arteries and vas deferens of guinea-pig were examined by freeze-etching. The most striking finding was that the surface vesicles lie in roughly longitudinal rows, with areas of membrane free of vesicles in between. The areas free of vesicles are believed to correspond to areas occupied by dense bodies in conventionally fixed and sectioned material. Other cell constituents which could be identified included sarcoplasmic reticulum and, probably, thick myofilaments.
The fine structure of the surface epithelium of the human ovary
Authors: Papadaki Lucienne , Beilby J. O. W.
Published: April 2021

The surface epithelium has been studied on human adult ovarian biopsies, one foetal ovary and an ovarian biopsy from a woman 12 weeks pregnant. In the adult, the surface of the ovary is covered by a single, patchy layer of epithelium which varies from squamous, through cuboidal to columnar in shape. Cytoplasmic projections extend into the peritoneal cavity from the free surface of the cells and their lateral borders are connected by desmosomes. The nucleus is irregular and the dense cytoplasm contains many polysomes, free ribosomes and tono-filaments. Intracellular vacuoles and extracellular channels contain materials of low to moderate density. Lipid droplets and compound aggregates are present and both coated and smooth vesicles occur in the peripheral cytoplasm. Focal degenerative changes may be seen which range from loss of density in the basal cytoplasm to complete desquamation.

In pregnancy the basal surface of the epithelium is deeply infolded with an increase in cytoplasmic lipid. In the foetal ovary the entire surface is covered with an epithelium.

From these studies and the work of others, it seems clear that, although the prime function of the ovarian surface epithelium is its contribution of pregranulosa cells during foetal development, many features remain which suggest that it has an important role in transport. It may also influence follicular development in the foetus and appears to have some potential for steroid metabolism. The patchiness and degenerative changes found in the adult human ovary may be related to fluctuations in hormonal balance.
The cytoplasmic incorporation of tritiated thymidine during oogenesis in Pteridium aquilinum
Authors: Sigee D. C. , Bell P. R.
Published: April 2021

The short-term incorporation of tritiated thymidine into the cytoplasm of cells undergoing oogenesis was investigated in Pteridium aquilinum using electron-microscope autoradiography. There was substantial uptake into the central cell and egg cell during the 6-h labelling period. The quantity and distribution of the label incorporated into the cytoplasm were closely similar in cells fixed immediately after the labelling period and in those immersed for a further 18 h in unlabelled thymidine. This suggested that incorporation was into a stable molecule, with little nucleoside turnover and no subsequent migration within the cytoplasm. Enzyme studies indicated that the tritiated thymidine was incorporated almost entirely into DNA, most probably the DNA undergoing replication.

Within the cytoplasm the label was markedly and consistently concentrated in plastids and mitochondria. This localization was not, however, complete and 5–40% was attributable to sites in the ground cytoplasm.

A gradient of incorporated label was demonstrated within the cytoplasm in both central cells and egg cells. Concentration was high adjacent to the nucleus and low at the cell periphery. This gradient could be satisfactorily explained by the distribution of the plastids and mito-chondria within the cytoplasm, the labelling of the organelles being irrespective of their position. The results of statistical examination of the frequencies of the silver grains associated with the mitochondria and plastids were considered to indicate general uptake of label directly into the DNA of these organelles without nuclear participation.
Electron stereoscopy as a means of classifying synaptic vesicles
Authors: Dennison Marion E.
Published: April 2021

In addition to the well known ‘round’ synaptic vesicles, 2 distinct types of ‘flat’ vesicle have been found by electron stereoscopy of aldehyde-fixed central nervous tissue. In the goldfish spinal cord presynaptic terminals are found which contain cylindrical vesicles, and others which contain disk-shaped vesicles. In the rat olfactory bulb no cylindrical vesicles are found; all the flat vesicles there appear to be disk-shaped.
The distribution of electron-dense tracers in peripheral nerve fibres
Authors: Hall Susan M. , Williams P. L.
Published: April 2021

Two electron-dense tracers, ferritin and lanthanum, have been administered to peripheral nerve fibres, and their uptake has been studied ultrastructurally. It was found that the peri-neurium was an effective barrier to ferritin in vivo, and the tracer was subsequently injected sub-perineurially. Ferritin uptake over a 120-min period was confined to occasional phagocytic vesicles in perineurial and Schwann cells, and to the nodal gap substance and paranodal peri-axonal space. No uptake was observed in the myelin sheath, incisural intraperiod line gap, or in the axoplasm. Soaking fibres in ferritin in vitro resulted in a more generalized cytoplasmic and axoplasmic uptake, although the myelin sheath and Schmidt-Lanterman incisures remained devoid of the tracer.

Lanthanum nitrate, included in the fixative solution, delineated the patent incisural intra-period line gap, and outlined the external surface of the terminal loops of nodal Schwann cell cytoplasm, and the paranodal Schwann cell-axolemmal junction. Unlike ferritin, La3+ penetrated the myelin sheath, being usually confined to the intraperiod line region of the outer lamellae, where it was associated with a widening of the lamellar unit, and an apparent splitting of the intraperiod line.

The results are discussed with regard to distribution of extracellular space in peripheral nerve fibres.
Immunochemical and ultrastructural mapping of the gelatin-binding and cellattachment regions of human plasma fibronectin with monoclonal antibodies
Authors: Hasty David L. , Courtney Harry S. , Simpson W. Andrew , Mcdonald John A. , Beachey Edwin H.
Published: April 2021

Monoclonal antibodies against fibronectin were used to locate the gelatin-binding and cell-attachment regions of plasma fibronectin at an ultrastructural level. A total of 23 hybridomas were generated using mice immunized with either intact fibronectin or a 40000 Mr gelatin-binding fibronectin fragment. One of these antibodies (D9b) strongly inhibited the interaction of radio-labelled fibronectin with gelatin. Another antibody (IB10) inhibited the attachment of Chinese hamster ovary (CHO) cells to a fibronectin substratum by 99%. Both of these antibodies were purified by affinity chromatography on columns of fibronectin-Sepharose and were then incubated with soluble fibronectin to form antigen-antibody complexes. The complexes were separated from free antibody on a column of Sephadex G-200 and were prepared for electron-microscopic examination by spraying on mica discs and rotary shadowing with platinum. As determined by this method, the fibronectin molecules measured 124 ± l.7nm in length. Monoclonal antibody IB10 was visualized as a globular projection 40 ± l.4nm from one end of the fibronectin filament. Monoclonal antibody D9b, on the other hand, was visualized as a globular projection at or near one or both ends of the molecule. These data provide the first morphological localization of the gelatin-binding and cell-attachment regions of fibronectin and indicate that further studies using mono-clonal antibodies directed toward other epitopes should shed light not only on function but also on the tertiary and quaternary structure of the fibronectin molecule.
Localization of low abundance dna sequences in tissue sections by in situ hybridization
Authors: Lo Cecilia W.
Published: April 2021

Specific DNA sequences were localized in the nuclei of paraffin-embedded mouse tissue sections with in situ hybridization using a biotinylated globin probe in conjunction with a streptavidin-alkaline phosphatase detection system. Globin inserts were clearly detected in the tissue sections of transgenic mice containing 1000, 120 or 5 copies of the exogenously introduced β-globin genes. In addition, specific hybridization signal was also obtained for the endogenous complement of ß-globin genes in the tissue sections of normal mice. These results demonstrate that this hybridization procedure is very sensitive and should be useful for characterizing the distribution of low abundance DNA sequences in cells and tissue sections.
Size uniformity of ribonucleoprotein matrix particles in loops of Pleurodeles waltlii lampbrush chromosomes visualized by electron microscopy
Authors: N’Da E. , Bonnanfant-Jaїs M. L. , Penrad-Mobayed M. , Angelier N.
Published: April 2021

The different kinds of loops of lampbrush chromosomes were identified in phase contrast, then analysed by electron microscopy on thin sections. Examination at high magnification showed that the basic structure of the ribonucleoprotein (RNP) matrix of all kinds of loops is a 30 nm RNP particle. Furthermore, this study suggests that the morphological differences between the loops are due to the extent of aggregation of these particles.
The extracellular electrical current pattern and its variability in vitellogenic Drosophila follicles
Authors: Bohrmann Johannes , Dorn Alfred , Sander Klaus , Gutzeit Herwig
Published: April 2021

We determined the extracellular electrical current pattern around Drosophila follicles at different developmental stages (7–14) with a vibrating probe. At most stages a characteristic pattern can be recognized: current leaves near the oocyte end of the follicle and enters at the nurse cells. Only at late vitellogenic stages was an inward-directed current located at the posterior pole of many follicles. Most striking was the observed heterogeneity both in current pattern and in current density between follicles of the same stage. Different media (changed osmolarity or pH, addition of cytoskeletal inhibitors or juvenile hormone) were tested for their effects on extrafollicular currents. The current density was consistently influenced by the osmolarity of the medium but not by the other parameters tested. Denuded nurse cells (follicular epithelium locally stripped off) show current influx, while an accidentally denuded oocyte produced no current. Our results show that individual follicles may be electrophysiologically different, though their uniform differentiation during vitellogenesis does not reflect such heterogeneity.
Transcriptive and replicative activity of the X chromosome in an autosomal segmental hyperploid in Drosophila and its significance
Authors: Ghosh Syamasri , Mukherjee A. S.
Published: April 2021

In the present investigation the transcription and replication patterns have been examined in different segments of the X chromosome and in certain specific segments (88B—92A) of an autosomal segmental hyperploid in which an extra segment 88B–92A (3R) is translocated to the X chromosome in addition to the normal two doses. Transcriptive activity monitored by [3H]uridine-labelling of these autosomal hyperploids reveals an enhanced hyperactivity of the male X chromosome while the female X chromosomes show no change in their activity. [3H]thymid1ne autoradiograms reveal that while the labelling frequencies of most replicating sites are distinctly lowered in the autosomal hyperploid males, no change within sexes is resolvable with regard to labelling-intensity profile. Furthermore, the X-autosome labelling frequency relation shows a distinct deviation from linearity, suggesting multiple events that lead to a higher template form of the X chromosome. These findings lead us to suggest that the signals emanating from auto-some(s) do not interfere with the primary modulation inherent in the X chromosome, but act on a modulated organization of the same at a second step evoking higher activity in the male X chromosome. The results further reveal that the gene activity of the X chromosome remains unaffected by the pattern of pairing of the autosomal segments.
Membrane fusion events in the Ca2+/ionophore-induced acrosome reaction of ram spermatozoa
Authors: Fléchon J. E. , Harrison R. A. P. , Fléchon B. , Escaig J.
Published: April 2021

An acrosome reaction was induced in ejaculated ram spermatozoa by treatment with calcium and the ionophore A23187. Samples were fixed at different times after initiation of induction, and the morphological changes within the head membranes that took place as exocytosis occurred were studied in freeze-fracture replicas.

Reacted acrosomes appeared in individual spermatozoa within the calcium/ionophore-treated population at different times after the start of treatment; the first cells had reacted by 10 min, whereas some took more than 40 min to react. No changes were observed in control populations.

An early effect of treatment (seen in most cells within 10 min) was the appearance of particle-free ‘clearings’ in the plasma membrane over the entire acrosomal region, with aggregation of intra-membranous particles between and around these ‘clearings’. At the same time, there was an increase in the number of large particles (⩾10 nm) within the plasma membrane over the ‘lunula’ of the equatorial segment and the anterior part of the post-acrosomal region.

Fusion of the plasma and outer acrosomal membranes began in a limited area at the border between the anterior and equatorial segments of the acrosome. It then spread, following arborescent pathways, sideways along this border and forwards towards the apex of the head. This labyrinthic propagation resulted in an ‘acrosomal cap’ increasingly fenestrated towards its posterior margin. Fusion propagation over the equatorial segment was inhibited, apparently as a result of the highly ordered structure of the membranes in this region.
Contacts of chick fibroblasts on glass: Results and limitations of quantitative interferometry
Authors: Bailey Juliet , Gingell David
Published: April 2021

We have examined the contacts made by explanted chick heart and limb bud fibroblasts after 24–48 h on glass, using quantitative interference reflection microscopy (IRM). Contacts beneath very thin cytoplasmic lamellae were avoided because the images of such contacts depend on the thickness of the lamellae.

Plaque-like focal contacts, distinguished on the basis of shape and low irradiance (darkness), are intimate adhesions to the substratum. These images can be interpreted if it is assumed that microfllaments associated with the lower membrane increase the local cytoplasmic refractive index. The range of irradiances measured for focal contacts was found to be rather wide, and our modelling shows that the most likely explanation for this is that the images receive variable contributions from the adjacent cytoskeleton. For this reason it is particularly difficult to assign a characteristic thickness for these contacts from IRM data.

Close contacts, seen principally as ‘grey’ regions under migrating cells at the edges of the expiants, also show a wide range of irradiances. Unlike focal contacts, it is not necessary to postulate any involvement of the cytoskeleton in their images and they can be modelled as regions where an aqueous glycocalyx zone about 20–30 nm thick separates the membrane bilayer from the glass. Paler grey regions that also look like close contacts are apparently formed where the cell surface has lifted several tens of nanometres from the glass.
Developmental and mitotic behaviour of two novel groups of nuclear envelope antigens of Drosophila melanogaster
Authors: Frasch Manfred , Paddy Michael , Saumweber Harald
Published: April 2021

Two novel groups of nuclear envelope antigens have been identified using monoclonal antibodies. On immunoblots the antigens correspond to distinct sets of polypeptides in the 175 ×103 molecular weight range. The antigens are enriched in a nuclear matrix-pore complex-lamina fraction of Drosophila tissue culture cells. We have studied the cellular distribution of these antigens throughout oogenesis and early embryo development.

Immunoblots show that one group of the 175 ×103Mr antigens is maternally transmitted to the embryo. This had already been observed for the 74/76× 103MrDrosophila lamins described previously, and we showed that a large proportion of the lamins is localized in the interior of the oocyte nucleus. We have also followed the fate of the high molecular weight antigens during mitosis. Each of the antigens uses a different pathway for its distribution to the daughter nuclei. These observations may give clues to the molecular mechanisms involved in the disassembly-reassembly process of the nuclear envelope.
Hyaluronic acid modulates proliferation of mouse dermal fibroblasts in culture
Authors: Yoneda Masahiko , Yamagata Masahito , Suzuki Sakaru , Kimata Koji
Published: April 2021

When the concentration of hyaluronic acid was monitored in primary cultures of mouse skin dermal fibroblasts, there was an increase in hyaluronic acid proportional to the increase in cell number during the logarithmic growth phase. The concentration reached the maximum value 2 days before the cells became confluent, and then decreased gradually. Hyaluronic acid added at 1 mg ml−1 during the logarithmic phase either promoted or inhibited cell growth, depending on the density of cells at the time when hyaluronic acid was added. Hyaluronic acid (1 mgml−1) added to subconfluent or postconfluent cultures induced a transient DNA synthesis with a consequent increase (>20%) in cell number. The effects appeared to be specific, since neither hyaluronic acid oligosaccharides nor some other types of glycosaminoglycan (chondroitin, chondroitin sulphates, heparan sulphates and heparin) had any similar effects. Dibutyryl adenosine 3′,5′-cyclic monophosphate (dbcAMP), at 1 mM, added to subconfluent or postconfluent cultures had promoting effects successively on hyaluronic acid synthesis and on cell growth. An increase in hyaluronic acid synthesis also occurred when dbcAMP was added to day 1 cultures in the logarithmic growth phase, but the effect on cell growth was reversed; there was an inhibition rather than a promotion. The pattern of cell density-dependent variation of the dbcAMP effect is quite similar to that observed with exogenously added hyaluronic acid. Therefore, we propose that hyaluronic acid added exogenously or supplied endogenously by increased synthesis may act as a modulator of mouse dermal fibroblast proliferation.
Hyaluronic acid-dependent change in the extracellular matrix of mouse dermal fibroblasts that is conducive to cell proliferation
Authors: Yoneda Masahiko , Shimizu Satoru , Nishi Yoshimi , Yamagata Masahito , Suzuki Sakaru , Kimata Koji
Published: April 2021

Fluorescein isothiocyanate (FITC)-labelled hyaluronic acid, when incubated with subconfluent cultures of mouse dermal fibroblasts, was incorporated into the extracellular matrix. Deposition of the fluorescent label reached the maximum about 48 h after its addition. Hyaluronic acid decasaccharide, but not octasaccharide, inhibited the incorporation of the fluorescent label, suggesting that at least 10 sugar units in length are necessary for the incorporation of hyaluronic acid. A 2M-urea extract of the cell layer had the ability to bind [3H]hyaluronic acid. Again, the binding was inhibited by hyaluronic acid decasaccharide but not by octasaccharide, suggesting the presence in the urea extract of a hyaluronic acid-binding molecule that may participate in the incorporation of hyaluronic acid. A supramolecular aggregate prepared by rate-zonal sedimentation from the 2M-urea extract contained chondroitin sulphate proteoglycans capable of interacting with hyaluronic acid. Their core molecules were identical in size with those from a hyaluronic acid-binding chondroitin sulphate proteoglycan (PG-M) previously described in chick embryo fibroblasts.

Immunofluorescence analyses with anti-proteoheparan sulphate antibodies indicated that both exogenous addition of hyaluronic acid and enhanced synthesis of hyaluronic acid caused a preferential decline in the proteoheparan sulphate level in the extracellular matrix. Subsequent to this change, the cells began transient DNA synthesis. We suggest that hyaluronic aciddependent modulation of the level of proteoheparan sulphate in the extracellular matrix could be a causal event of cell proliferation.
The rescue of oral development of defective-micronucleate conjugants of Paramecium tetraurelia by normal gametic nuclei
Authors: Chau M. F. , Ng Stephen F.
Published: April 2021

The present study further analyses the importance of postmeiotic divisional derivatives of the micronucleus in the development of the oral apparatus of Paramecium during sexual reproduction. Cell lines possessing defective micronuclei generated by laser microbeam irradiation of the micronucleus were employed. They exhibited anomalies in nuclear reorganization and stomatogenesis in the sexual cycle. During autogamy, in some cells the micronuclear cycle terminated shortly after meiosis, resulting in the loss of all postmeiotic micronuclear derivatives. Stomatogenesis became arrested at an early stage of assembly of the oral membranelles, but the old oral apparatus was resorbed as usual, leading to the production of astomatous cells at the end of the sexual cycle. Conjugation of these cell lines with normal micronucleates rescued both nucleogenesis and stomatogenesis in the defective-micronucleate conjugant, primarily as a result of transfer of the male gametic nucleus from the normal conjugant to the defective-micronucleate mate. These observations demonstrate the stomatogenic significance, in particular in the initiation of oral membranelle assembly, of the gametic nuclei during sexual reproduction. The present study also suggests the possibility of micronuclear activities in the early part of the sexual cycle affecting postzygotic nucleogenesis.
Partition of protein (mass) to sister cell pairs at mitosis: A re-evaluation
Authors: Sennerstam Roland
Published: April 2021

Since the 1960s it has been thought that there is to some extent a difference in the partition of mass to daughter cells at mitosis. Recent studies using modern techniques give further support to such a phenomenon, which has become almost an axiom in cell biology. It has been suggested that such unequal distribution of metabolic constituents at mitosis contributes to the dispersion in cell generation times.

In the present work, PCC3 embryonal carcinoma (EC) cells were studied as undifferentiated G1 sister pairs by microspectrophotometry (MSP) following Feulgen-Naphthol Yellow staining (FNYS), in order to evaluate their protein content. Despite the considerable intraclonal intermitotic time heterogeneity found in undifferentiated PCC3 EC cells, it was concluded that the postmitotic difference in mass (protein) between sister cell pairs exerts a minimal influence upon the cell population mass variability, whereas it was deduced to have an influence upon variation in interphase time duration when comparing sister cell pairs. This offers a cell-physiological explanation to the randomly distributed difference repeatedly found between sister cell generation times. Furthermore, there was no correlation seen between the mass difference found between sister cell pairs postmitotically and the size of the mother cell.
Newt epidermal cell migration over collagen and fibronectin involves different mechanisms
Authors: Donaldson Donald J. , Mahan James T. , Smith Gerald N.
Published: April 2021

Effects of the synthetic peptides, Arg-Gly-Asp-Ser (RGDS), the amino acid sequence representing the fibroblast attachment site in fibronectin, and Arg-Gly-Glu-Ser (RGES), on collagen- and fibronectin-mediated migration in newt epidermal cells were compared. When RGDS at 50 μg ml− 1 was included in the incubation medium of skin explants, migration in fibronectin-coated dishes was almost totally blocked. In type I collagencoated dishes, this concentration of RGDS also inhibited migration, but to a lesser degree than on fibronectin. With 250 μg ml− 1 of RGES in the medium, the reverse was true. Here, migration on collagen was practically non-existent, while migration on fibronectin was affected only moderately. Collagen-mediated migration was sensitive to RGDS even when the peptide was added after migration on the coated substratum was well underway.

At a coating concentration of 10 μg ml−1 CB3, a cyanogen bromide fragment of the collagen alpha 1(1) chain, which contains no RGD sequences, was as good a migration substratum as intact collagen applied at the same coating concentation. At lower concentrations intact collagen was somewhat better than equivalent concentrations of CB3. The presence of RGDS in the medium throughout an experiment inhibited migration in CB3-coated dishes in a manner similar to its effect in dishes coated with collagen. On both substrata there appeared to be a peptidesensitive and a peptide-insensitive component to migration. The inhibitory effect of RGES on CBS-mediated migration was also similar to its effect in collagen-coated dishes.

Monensin, an ionophore known to block secretion and endocytosis inhibited migration on fibronectin and collagen to a small, but statistically insignificant degree. A more pronounced, statistically significant effect was seen in CBScoated dishes. Electrophoretic analysis of the cell extract and the medium of cells migrating overnight on CB3 in the presence of [35S] methionine showed no evidence of fibronectin secretion by the migrating cells.

These results indicate that newt epidermal cells use different mechanisms as they migrate over fibronectin and collagen. The data could be explained by the existence of two classes of extracellular matrix receptors on epidermal cells or one class of receptor that binds to the active site(s) in fibronectin and collagen with different affinities. Our inability to detect fibronectin secretion by cells migrating on CB3 and the collagen-like sensitivity of CB3-mediated migration to RGDS suggests that some epidermal receptors involved in migration are capable of recognizing more than just the RGD sequence. The effect of monensin in CB3-coated dishes may be an indication that receptor recycling occurs during migration over some substrata.
The molecular biology of receptors. techniques and applications of receptor research
Authors: Hughes R. C. , Strosberg. A. D.
Published: April 2021

The stated aim of this book is to present relatively briefly the main technical approaches being used for the characterization of cellular receptors involved in recognition of externally applied biologically active ligands. The emphasis is on the chemical isolation and characterization of receptors and there is little discussion of signal transmission and subsequent cell activation. The research topic is huge, is growing very rapidly and is a favoured target for many comprehensive treatments.
A gentle method for preparing cyto- and nucleo-skeletons and associated chromatin
Authors: Jackson D. A. , Yuan J. , Cook P. R.
Published: April 2021

We describe a method for permeabilizing and extracting cells that preserves both structure and function whilst allowing the cell derivatives to be handled freely. Cells are encapsulated in microbeads of agarose; the coat of agarose, which is freely permeable to small molecules, forms a protective layer around fragile cell constituents. Cells are then permeabilized by the non-ionic detergent Triton X-100 or antibody and complement in a buffer whose ionic composition mimics that of the cytoplasm. The resulting structures have been characterized morphologically (by immunofluorescence and electron microscopy) and biochemically. Lysis with Triton removes both cell and nuclear membranes, and extracts most of the cytoplasm to leave chromatin surrounded by cytoskeleton; nucleus and cytoplasm then become accessible to triphosphates, enzymes and antibodies. Lysis with complement permeabilizes the cell membrane but leaves the nuclear membrane intact; triphosphates and restriction enzymes, but not antibodies, can then enter both nucleus and cytoplasm. Both types of lysis yield preparations whose chromatin template remains essentially intact, and which is replicated and transcribed at rates close to, or greater than, those found in vivo. Treatment of complement-lysed cells with Triton reduces the very efficient DNA synthesis, implying that the nuclear membrane is involved, directly or indirectly, in replication.
The role of the RAS oncogene in the formation of tumours
Authors: Gilbert Penny X. , Harris Henry
Published: April 2021

A c-Ha-ras 1 oncogene, cloned from the EJ human bladder carcinoma cell line, was inserted into a shuttle vector carrying the selectable marker gene gpt that encodes the enzyme xanthine-guanine phosphoribosyl transferase. This construct, pSV2gptEJ, was transfected into NIH 3T3 cells by the calcium phosphate precipitation method and cells that had incorporated the plasmid were selected by growth in the presence of mycophenolic acid to which gpt confers resistance. A number of transfectant clones were tested for tumorigenicity by inoculation into nude mice. The take incidence was variable and the tumours arose only after a prolonged latent period. Many inocula produced no tumours. These results were consistent with the view that the tumours arose by selective overgrowth of minority cell populations.

Cell lines were derived by explantation of these tumours and were back-selected in 2-thioxanthine, a cytotoxic analogue of the xanthine-guanine phosphoribosyl transferase substrate. Five clones were obtained that did not express detectable levels of the c-Ha-ras 1 oncogene product, p21ras. All of them showed a less-transformed morphology than the transfected NIH 3T3 cells from which they originated. Nonetheless three of these clones were found to be tumorigenic at all sites tested. This finding demonstrates that once tumorigenic variants have been selected from the ras-transformed cells, continued production of the p2lras protein is not necessary for the maintenance of tumorigenicity.

Cytogenetic analysis revealed that the transfection procedure itself introduced major and stable perturbations of the genome of the transfected cells and confirmed that tumours were produced by selective overgrowth of variants with a chromosome constitution palpably different from that of the majority of the cells injected. In the light of the complex background of genomic changes produced in NIH 3T3 cells by transfection with the c-Ha-ras 1 oncogene, no conclusion can be drawn in genetic terms concerning its dominance.
Fracture-Flip: New High-Resolution Images Of Cell Surfaces After Carbon Stabilization Of Freeze-Fractured Membranes
Authors: Forsman Catarina Andersson , Silva Pedro Pinto Da
Published: April 2021

We introduce fracture-flip, a method of obtaining high-resolution views of membrane surfaces. The method, a corollary of label-fracture, is based on the stabilization of the exoplasmic halves of membranes by carbon evaporation. Inversion of these casts followed by Pt/C imaging leads to new views of the cell surface at macromolecular resolution.
Caffeine induces uncoordinated expression of cell cycle functions after ultraviolet irradiation: Accelerated cycle transit, sister chromatid exchanges and premature chromosome condensation in a transformed indian muntjac cell line
Authors: Musk S. R. R. , Downes C. S. , Johnson R. T.
Published: April 2021

Caffeine enhances the lethal effect of DNA-damaging agents. It also affects the timing of events in the cell cycle; the enhanced cytotoxicity may be partly due to caffeine’s ability to over-come the protective damage-induced delay in S or G2 phase. When the effects of caffeine are compared in a normal Indian muntjac cell line and a simian virus 40 (SV40)-transformed, ultraviolet light (u.v.)-sensitive line in which u.v. induces many sister chromatid exchanges, different cell cycle sensitivities are seen.

In the SV40-transformed line, caffeine over-rides the delay in DNA synthesis imposed by DNA damage; it initiates late cycle events after u.v. irradiation, and in some cases it induces S-phase premature chromosome condensation, apparently by inducing mitotic factors in cells where the chromatin is still replicating. Caffeine hasbeen reported to induce similar premature chromosome condensation in cells arrested with DNA polymerase inhibitors; this is the first recognition that such events occur in response to caffeine treatment after DNA damage. Some apparent paradoxes of caffeine’s action on irradiated cells can be thus explained.

In the normal line, caffeine neither affects progression through the cycle nor induces premature chromosome condensation after irradiation. In both lines, caffeine increases killing and sister chromatid exchanges after u.v. irradiation; but these effects are more pronounced when exerted through the cell cycle-related mechanism.
DNase I Nick Translation In Situ On Meiotic Chromosomes Of The Mouse, Mus Musculus*
Authors: Raman Rajiva , Singh A. P. , Nanda Indrajit
Published: April 2021

DNase-I-sensitive sites have been located on the meiotic chromosomes of the mouse, Mus musculus, by the in situ DNase I nick-translation method. We find that: (1) of all the cell types studied, pachytene nuclei are the most sensitive to DNase I; (2) in diplotene the nicks occur preferentially in the vicinity of chiasmata; (3) the sex chromosomes are also sensitive to the enzyme despite their transcriptional quiescence; and (4) in the sex bivalent the nicks are primarily observed in the putative region of recombination. We conclude that, in addition to discriminating between the transcriptionally active and inactive states of chromatin, DNase I identifies recombination-specific chromatin changes in meiotic prophase.
Chondrons from articular cartilage: I. Immunolocalization of type vi collagen in the pericellular capsule of isolated canine tibial chondrons
Authors: Poole C. Anthony , Ayad Shirley , Schofield Jennifer R.
Published: April 2021

A heterogeneous population of intact chondrons extracted from low-speed homogenates of canine tibial cartilage were stained by indirect immune-fluorescence methods with a polyclonal antibody to type VI collagen. In each of the four chondron groups examined, anti-(type VI collagen) anti-serum was concentrated in the capsule immediately adjacent to the chondrocyte complex. A constant but weaker fluorescent reaction persists in ‘tail-like’ extensions common to single and double chondrons and in the medial connections between adjacent chondrons in linear columns and aggregated clusters. Frayed collagen bundles typical of chondron preparations did not react with the antibody. Similarly, chondrons reacted with normal rabbit serum, or treated by type VI collagen extraction procedures, showed no staining reaction. The differential localization of type VI collagen in the pericellular capsule is discussed in relation to the maintenance of the chondron’s integrity and to the protection of the chondrocyte during dynamic compressive loading.
Diacylglycerol-Induced Shape Changes, Movements And Altered F-Actin Distribution In Human Neutrophils
Authors: Zimmermann A. , Gehr P. , Keller H. U.
Published: April 2021

The study shows that diacylglycerols (DAGs) as physiological activators of protein kinase C induce characteristic shape changes in human neutrophil granulocytes. In contrast to chemotactic peptides, which can induce front-tail polarity characterized by a contracted tail and an expanding front, DAGs elicit the formation of non-polar cells with surface projections. These cells exhibit a distinct type of motility characterized by vigorous and continuous shape changes without front-tail polarity and without the unidirectional movement and cytoplasmic streaming seen in polarized cells. In neutrophils exposed to DAGs, F-actin is shifted to the cell periphery and mainly into the surface projections of activated cells. DAGs induce the formation of large intracellular vacuoles in neutrophils producing surface projections, and these vacuoles persist after the cells have reacquired a spherical shape. Combinedstimulation of human neutrophils with DAG and fNLPNTL results in a suppression of peptide-induced polarity and the formation of non-polar motile cells resembling those stimulated with DAG alone. These results suggest that the diacyl-glycerol-protein kinase C pathway may be in-strumental in transducing or modulating signals to both the locomotor apparatus and the exocytotic and/or pinocytic system of the cell. Neutrophil stimulation with DAGs thus represents a useful model with which to study further the hypothesis that distinct types of neutrophil shapes and movements are preferentially associated with distinct functions and to characterize signalling pathways.
Contact Guidance On Microfabricated Substrata: The Response Of Teleost Fin Mesenchyme Cells To Repeating Topographical Patterns
Authors: Wood Andrew
Published: April 2021

The mesenchymal tissue component of the teleost pectoral fin bud has been explanted onto microfabricated quartz discs containing patterns of regular grooves of dimensions similar to those encountered by contact-guided cells in the intact fin system. Computer-aided analysis of cell migration on five separate patterns, from 1·8 to 7·4 μm repeat spacing, revealed that migration was predominantly aligned parallel to the long axis of the grooves, with individual cells becoming highly polarized, and the highest index of alignment (3·7) produced on the widest repeat spacings. When these cells are cultured on planar quartz, which is both chemically and physically isotropic, they flatten and adopt a morphology similar to that shown by cultured fibroblastic cell types on flat surfaces, suggesting that fin mesenchymal cells are not intrinsically predisposed to adopt an arborized morphology, like that observed in the intact fin bud. These results clearly demonstrate a directional migration in response to substratum topography, from a cell type known to be contact guided in vivo, and in the complete absence of adhesive heterogeneities.
Cell surface heparan sulphate and adhesive property of sublines of rat ascites hepatoma AH7974
Authors: Kimura A. , Kawaguchi T. , Ono T. , Sakuma A. , Yokoya Y. , Kochi H. , Nakamura K.
Published: April 2021

Two variants (74AD and 74FL) established from rat ascites hepatoma AH7974 were examined for the production of glycosaminoglycans in culture.

There was no difference between the adhesive (74AD) and the floating (74FL) variants in quantity of glycosaminoglycans produced by their cultivation in minimum essential medium supplemented with 10% foetal calf serum. However, they were distinctly different in the distribution patterns of heparan sulphate. In 74FL, about 70% of total heparan sulphate was found in the culture medium in soluble form, whereas in 74AD, only 7 % was found in the medium and the rest was in the cell-substratum complex.

In a serum-free medium, 74AD cells grew with-out adhering to the substratum. After cultivation, more than 90% of total heparan sulphate was found in the cell-associated fractions and the rest in the substratum fractions. No heparan sulphate was detected in the culture medium. On the other hand, 74FL cells released heparan sulphate to the serum-free medium as much as to the serum-containing medium.

The increase in amount of heparan sulphate in the culture medium of 74FL cells was supposed to be caused by failure of the cells to deposit heparan sulphate at the cell surface and not caused by increased production.

Cell-substratum adhesion mechanisms involving cell surface heparan sulphate (heparan sulphate proteoglycan) and some serum intermediate(s)) are discussed for 74AD cells.
Effects of suboptimal levels of extracellular calcium on the regulation of the cyclic amp phosphodiesterase-inhibitor system and membrane differentiation in Dictyostelium Discoideum
Authors: Cameron A. M.
Published: April 2021

When starved wild-type amoebae of Dictyo-stelium discoideum were washed and incubated in 1 mM-EGTA, their ability to induce soluble cyclic AMP phosphodiesterase (PD) activity in response to either millimolar cyclic AMP or a series of nanomolar cyclic AMP pulses was reduced by 55–75 %. Supplementation of EGTA-treated cells with exogenous Ca2+ stimulated PD induction in a dose-dependent fashion (ECS0 = 100–200 nM free extracellular Ca2+), and enzyme production was maximal at about 1μM free Ca2+. Ca2+ depletion also strongly impaired production of the phosphodiesterase inhibitor (PDI). In contrast, other than delaying their appearance by about 1 h, EGTA had little effect on the induction by cyclic AMP pulses of cell surface markers such as contact sites A and membrane-bound PD activity. Similar changes in both the soluble and membrane activities were observed with strain NP368, a mutant that overproduces cyclic GMP when stimulated by cyclic AMP. Thus, Ca2+ depletion does not appear to inhibit PD and PDI production by reducing intracellular cyclic GMP. To determine whether Ca2+ depletion alters signal transduction, two mutants that produce the soluble PD activities constitutively were examined. Suboptimal concentrations of free extracellular Ca2+ were found to inhibit PD production in these cells to the same degree and with the same concentration dependence as low Ca2+ inhibited PD induction by cyclic AMP in wild-type cells. These results suggest that Ca2+ depletion by EGTA probably inhibits PD and PDI production indirectly by perturbing an intracellular Ca2+ pool(s) rather than by altering a surface cyclic AMP-receptor-mediated process.
Molecular organization of the cell wall and cell-wall crystals from Chlamydomonas eugamatos
Authors: Goodenough Ursula W. , Heuser John E.
Published: April 2021

The unicellular green alga Chlamydomonas eugamatos is surrounded by a glycoproteinaceous cell wall. Using the quick-freeze deep-etch technique, we analyse the organization of the wall layers in situ, and delineate the structure of the outermost crystalline layer (W6). We also show the structure of the protein monomers, and propose a model for their organization in the crystalthat is similar to that deduced for the related Chlorogonium elongatum crystal by image reconstruction analysis. We also show that the C. eugamatos crystal is highly similar to the flagellar collar of Chlamydomonas reinhardtii.
Microtubule-dependent cell cycle regulation is implicated in the G2 phase of Hydra cells
Authors: Dübel Stefan , Little Melvyn
Published: April 2021

Interstitial cells of Hydra attenuata, from which nerve cells and nematocytes (stinging cells) differentiate, were arrested in either metaphase or G2 by different concentrations of the microtubule-depolymerizing agent nocodazole. At a concentration of 1 ·4 nM-nocodazole, a large number of cells were arrested in metaphase. However, at concentrations of 2 nM-nocodazole and above most of the cells were arrested at a distinct point in G2 several hours before mitosis. After removal of the 2nM-nocod-azole block, 75% of the cells entered the next cell cycle about 10 h later. To our knowledge this is the first time that cells have been synchronized by arresting them in the G2 phase. Visualization of Hydra microtubules with a tubulin monoclonal antibody and immunofluorescent staining showed that the very low concentrations of nocodazole used for cell cycle arrest were indeed affecting microtubule structures. Spindles and stem cell microtubules disappeared at 0 ·;8-1 nM-nocodazole, followed by nerve microtubules (about 2 nM), cnidocil microtubules (10 nM) and finally by nematocyte microtubules (34 nM). Taken together, these data strongly indicate a microtubule-dependent mechanism of cell cycle regulation in the G2 phase.
Inhibition of stenotele commitment by an endogenous factor in Hydra
Authors: Fujisawa Toshitaka
Published: April 2021

The precise timing of the inhibition of stenotele differentiation by an endogenous factor in Hydra was localized in the terminal cell cycle of the precursor cells. Animals were treated with crude extract of Hydra and the inhibition kinetics of stenotele differentiation were determined. Comparison of these results with the differentiation kinetics of stenoteles determined by using [3H] thymidine labelling indicated that an endogenous factor inhibited precursor cells from entering the stenotele pathway near the S/G2 boundary of the terminal cell cycle. Since stenotele commitment occurs during this time, these results suggest that an endogenous factor specifically blocks stenotele commitment. Precursor cells blocked by the factor appeared to choose other pathways.