bluuebunny/crossref_metadata_2025 huggingface dataset:
Tracheid formation in cultures of pine (Pinus sylvestris)
Authors: RAMSDEN L. , NORTHCOTE D. H.
Published: April 2021

Various combinations of growth factors were tested for their ability to induce phenylalanine ammonia lyase activity in a suspension culture of Pinus sylvestris L. Phenylalanine ammonia lyase is an established marker of differentiation in angiosperm tissue culture. A growth medium capable of inducing phenylalanine ammonia lyase activity was developed; it contained basal medium supplemented with 10mg 1−1 (53.5 μM) naphthaleneacetic acid, 2mgl−1 (9.3 μM) kinetin and 6% (175mM) sucrose. Using this medium it was possible to induce the differentiation of tracheids after 25 days in culture. Secondary thickening of the walls of cells cultured in the induction medium was confirmed by microscopical examination and correlated with an increase in the lignin content and changes in the polysacchar-ide composition of the cell wall. After culture in the induction medium there was an appreciable increase in the mannose content of wall polysaccharides, which suggested that synthesis of secondary wall glucomannan had been induced by the change in growth factor concentrations. High levels of xylose present in the cultured cells suggested the presence of a xyloglucan, which was subsequently isolated and characterized. 13C n.m.r. (nuclear magnetic resonance) spectroscopy was used to identify the glycosyl linkages in the xyloglucan.
Surface events in polymorphonuclear leucocyte activation: insights from a hydrophobic membrane antigen that triggers the respiratory burst
Authors: Rosen H. , Gordon S.
Published: April 2021

The rat monoclonal antibodies (Mab) 1A10.4 and IG4 were raised to a solubilized NADPH-oxidase preparation from guinea-pig polymorphonuclear neutrophils (PMN). They bind to a surface antigen restricted to guinea-pig myelomonocytic cells and on binding to the PMN surface can stimulate the respiratory burst (RB) and degranulation. This response was specific to these Mab and was not found with several other Mab restricted to myelomonocytic cells but with other antigenic epitopes. In order to understand the role of this molecule in stimulating the RB, we have characterized the antigen (Ag) by a variety of techniques. It is a hydrophobic membrane molecule with an apparent molecular weight of 8–10 (× 103) and a pI of 6·2. The Ag has been partially purified by extraction in organic solvents and high-pressure liquid chromatography on silica and is probably a proteolipid. Studies on Mab-dependent triggering of PMN secretion indicated that cross linking of the cell surface was critical. We therefore used direct immunofluorescence under triggering and non-triggering conditions to show that stimulation of the RB by Mab correlated with redistribution of the surface Ag into patches. This patching was associated with aggregation of surface Ag and transfer of Ag from a Triton X-100-extractable to a Triton X-100-inextractable membrane domain. These movements of surface Ag, which included both patching and capping resulting from aggregation of a hydrophobic membrane component, occurred at 4°C and were insensitive to inhibition by cytoskeletal inhibitors. These specific probes that control triggering of the RB have permitted the dissection of PMN stimulation into discrete membrane events by correlating the biochemical and morphological characteristics of a new PMN surface Ag as it stimulates exocytosis.
Coumarin–phalloidin: A new actin probe permitting triple immunofluorescence microscopy of the cytoskeleton
Authors: Small J. V. , Zobeley S. , Rinnerthaler G. , Faulstich H.
Published: April 2021

7-Diethylamino-3-(4-isothiocyanotophenyl)-4-methylcoumarin (CPITC) was coupled to amino-methyldithiolanophalloidin to produce a new phalloidin derivative, coumarin–phalloidin, fluorescent in the blue region of the spectrum. Coumarin–phalloidin binds to actin with around 100-fold less affinity than unconjugated phalloidin, but with enough avidity to make it a useful stain for actin filaments. Appropriate filter combinations permit triple immunofluorescence microscopy of the cytoskeleton with fluorescein and rhodamine conjugates together with coumarin-phalloidin.
Review: Video-enhanced light microscopy and its applications in cell biology
Authors: Shotton David M.
Published: April 2021

The combination of novel optical microscopic techniques with advanced video and digital image-processing technology now permits dramatic improvements in the quality of light-microscope images. Such video-enhanced light microscopy has lead to a renaissance in the applications of the light microscope for the study of living cells in two important areas: the intensification of faint fluorescence images, permitting observation of fluorescently labelled cells under conditions of very low illuminating intensity; and the enhancement of extremely low contrast images generated by minute cellular structures, so that these may be clearly seen and their normal intracellular movements recorded.

Application of both these aspects of video-enhanced light microscopy have recently led to major discoveries concerning the functioning of the living cell. In this review I discuss the equipment, procedures and image-processing principles employed in these applications, and describe and illustrate some of the spectacular results that have recently been obtained.
Clonality of preneoplastic liver lesions: Histological analysis in chimeric rats
Authors: Weinberg W. C. , Lannaccone P. M.
Published: April 2021

The clonality of chemically induced altered hepatocellular foci was examined in rat liver. Chimeric rats composed of two histologically distinguishable cell lineages were placed on an initiation-promotion protocol for liver cancer induction. This resulted in multiple lesions of altered enzyme expression. These altered hepatocellular foci are generally considered to be initiated sites susceptible to cancer formation. The cellular origins of these lesions were determined by aligning sections demonstrating cell lineage with serial sections stained for altered enzyme expression. Analysis included 110 areas of deficient ATPase (EC 3.6.1.3) activity and 59 glucose-6-phosphatase (EC 3.1.3.9; G-6-Pase) deficient lesions, 744 foci of re-expression of γ- glutamyl transpeptidase (EC 2.3.2.2; γ-GT), and decreased glycogen mobilization (187 lesions). of the 1100 focal enzyme alterations, 1054 were shown to be composed entirely of cells from a single lineage of the two lineages present in the mosaic tissue. Multiple alterations occurred within given lesions. Lesions with up to four phenotypic alterations were found to consist of cells of a single lineage. These results suggest that individual enzyme-altered foci are clonal in origin and that phenotypic heterogeneity within altered hepatocellular foci is due to lesion progression within a clonal population and not to a multicellular derivation.
The preservation of specialization by dispersed steer thyroidal cells in long-term culture
Authors: Siegel E.
Published: April 2021

Serially propagated cells derived from the steer thyroid gland preserve several specialized characteristics, some demonstrable for as long as 8 months (over 15 passes). For about 5 passes (3 months), follicular-like arrays of cells develop. Thyroid-stimulating hormone (TSH) or thyrotropin (1 and 10 mu./ml) induces the formation of supernumerary nucleoli, and promotes at the ultrastructural level the prompt (within 10 min) appearance of microvilli, pseudopodia, and intracytoplasmic droplets. These cells have a mean plating efficiency (PE) of 16·5 ±4·5 % (standard deviation) and with 3 × 104 cells as the standard inoculum, a mean doubling time (Td) of 43·7± 1·2 h. The linear variation of Td with the number of cells seeded probably signifies strong cell-cell interactions. TSH (1–50 mu./ml) influences both PE and Td, with 1 mu./ml producing the maximum stimulation. TSH (0·001–100 mu./ml) also induces the discharge of incorporated radio-iodide from the cultured thyroid cells, achieving a peak by 2–4 h, titres of 0·1–1 mu./ml being most potent. Long-acting thyroid stimulator (LATS) (0·2 u./ml) likewise effects release of 131I into growth medium (1 experiment), but over a more protracted period. After labelling with [3H]leucine, radioautographs demonstrate that the synthesis of proteins is stimulated by exposure to TSH (1 mu./ml). Precipitation with trichloro acetic acid indicates that these cells persist in synthesizing 131I-tagged iodoproteins, an activity optimally stimulated by 1 mu./ml TSH and marked by 3 h. Hence, such thyroidal cell lines afford a useful model for studying differentiation and hormonal effects.
The toxic effect of spermidine on normal and transformed cells
Authors: Otsuka H.
Published: April 2021

The cytotoxic effect of spermidine was investigated on various tissue culture cell lines in the presence of calf serum. Cells incubated with cytotoxic concentrations remained rounded up while cells in control cultures always spread out on the surface of the Petri dish. The spermidine concentration tolerated depends on cell number, concentration of serum, the strain of cells used and the phase of the growth cycle.

The spermidine index (SI) of a cell culture is defined as the highest level of spermidine which did not show cytotoxic effect in the standard test system. The SI measures the ability of a cell culture (or line) to neutralize the cytotoxic effect of spermidine.

The SI of normal fibroblastic cells such as BHK21/C13 or mouse embryo cells alters characteristically with different phases of the cell cycle. It is highest in lag or early exponential phase, then it diminishes during the exponential growth phase reaching its lowest point after the cell culture has become confluent.

It is characteristic of polyoma virus-transformed cell lines and of other established permanent cell lines (which are probably of spontaneously transformed origin) that their SI decreases only slightly at high cell densities. There is a correlation between higher SI of transformed cells and their ability to grow in soft agar suspension.
The ultrastructure of fertilization and zygote formation in the green alga Ulva mutabilis føyn
Authors: Braten T.
Published: April 2021

Fertilization and zygote formation in the multicellular green alga Ulva mutabilis Føyn were studied with the aid of transmission and scanning electron microscopes. The growth mutant ‘slender’ was used in this investigation. The initial contact between mating gametes is made by the flagella, whereupon cytoplasmic contact is established within a few seconds. Fusion of the cytoplasm of the 2 cells is completed in 5 min and the nuclei can be seen to fuse 30 min after the onset of copulation. The settling of the young zygotes initiates a process during which the 4 flagella are absorbed into the cell. Cell wall formation also starts when the zygotes settle. Observations indicate disintegration of one of the 2 chloroplasts in the young zygote.
The response of haemopoietic cells to growth factors: developmental implications of synergistic interactions
Authors: Heyworth Clare M. , Ponting Ian L. O. , Dexter T. Michael
Published: April 2021

Haemopoietic cell growth factors are normally assayed using unfractionated marrow cells (NBM). However, using this population it is difficult to distinguish between direct versus indirect effects, because of the low incidence of colony forming cells (CFC) and the presence of possible accessory cells (which may themselves be acted upon by the growth factors and stimulated to produce other growth stimulatory or inhibitory molecules that influence the development of the CFC). Furthermore, NBM contain the whole spectrum of multipotent and lineage-restricted CFC and it is often difficult to determine precisely which populations are being stimulated to develop. This latter problem can be solved, in part, by using marrow from mice previously treated with 5-fluorouracil (5-FU): an agent that preferentially kills the more mature, actively cycling CFC but spares the proliferatively quiescent multipotent stem cells. Since the 5-FU-treated marrow also contains many possible accessory cells, however, it is again not clear whether or not the responses elicited by growth factors are due to direct or indirect effects upon the CFC. To circumvent this problem we have obtained a highly enriched population of multipotent stem cells (FACS-BM) that is free of accessory cells, and have compared the responses of these cells to NBM and to 5-FU-BM in the presence of a variety of growth factors. The data demonstrate that interleukin-1 (IL-1), which is not a growth factor by itself, can act synergistically with other growth factors on FACS-BM and 5-FU-BM but not on NBM. With FACS-BM, IL-1 can synergize with GM-CSF and with M-CSF but not with IL-3 or G-CSF. With 5-FU-BM, IL-1 can synergize with GM-CSF, M-CSF and IL-3. Furthermore, G-CSF, a poor growth stimulus when used alone, can synergize with GM-CSF and M-CSF, using either FACS-BM or 5-FU-BM, and can also synergize with IL-3 when using 5-FU-BM but not with FACS-BM. The data further indicate that extensive overlap occurs between the multipotential cells that respond to IL-3 alone and those that can respond to a combination of other growth-promoting stimuli such as IL-1 plus CSF-1 or G-CSF and CSF-1. This indicates that at least some multipotential cells have receptors for all the growth factors used in this study, but that combinations of growth factors sometimes need to be present in order to facilitate proliferation and development. Furthermore, the outcome of the response in terms of mature cell lineages produced is a reflection of the stimuli to which the multipotential cells are exposed. The developmental implications of these findings are discussed.
Glycosaminoglycan expression in intestinal epithelial skin-fibroblastic cell cocultures: Fibroblastic cell-mediated effects of glucocortlcoids
Authors: Bouziges Françoise , Simon-Assmann Patricia , Leberquier Cathy , Haffen Katy , Kedinger Michèle
Published: April 2021

The nature and distribution of newly synthesized glycosaminoglycans (GAGs) were studied in foetal rat skin fibroblasts, in rat intestinal endodermal cells and in cocultures of both cell types. The data show that fibroblasts synthesize and secrete hyaluronic acid (HA), heparan sulphate (HS) and chondroitin sulphate molecules (CS). Our data focus on HA, which is found as two different molecular forms, the smallest hydrodynamic-sized species being mostly recovered within the cell or associated with the cell surface, and the largest one secreted into the medium, whatever the cell type. Endodermal cells synthesize only two types of GAGs: the low molecular weight form of HA and HS.

Cocultures of rat intestinal endodermal and skin fibroblastic cells in the presence of dexamethasone (Dx), allow optimal epithelial cytodifferentiation (Kedinger et al. 1987a). The main changes in the GAGs synthesized under these conditions as compared to skin fibroblastic cell cultures concern: (1) the enhancement of the lowest molecular weight form of HA to the detriment of the highest form in the cellular, pericellular and extracellular compartments; (2) the increase in the proportion of HS molecules associated with the cell surface. Interestingly, similar modifications are obtained by addition of Dx to the skin fibroblastic cell cultures.

The data are discussed with reference to the constitution of a basement membrane at the epithelial-fibroblast interface in the cocultures, to the fibroblastic-dependent induction of epithelial differentiation and to the glucocorticoid response.
Scanning electron microscopy of focal contacts on the substratum attachment surface of fibroblasts adherent to fibronectin
Authors: Singer Irwin I. , Kazazis Diana M. , Scott Solomon
Published: April 2021

We have examined the cell-to-substratum attachment surface of hamster fibroblasts with scanning EM, and describe the surface ultrastructure of focal contacts and microspikes during cellular attachment and spreading on fibronectin. Nil 8 fibroblasts were seeded onto fibronectin-coated glass coverslips in serum-free medium, fixed, and the fibro-blast-fibronectin monolayer was separated from the glass and inverted for scanning electron microscopic (EM) analysis. Focal contact development was detected by interference reflection microscopy and correlated with the immunofluorescence microscopic distribution of fibronectin receptor antigens. The cell undersurface appeared smooth and featureless at 0·5 h when focal contacts were undetectable and fibronectin receptors were distributed diffusely. By 1–2 h, undersurface membrane impressions of focal contacts were detected with scanning EM; their size, shape and distribution matched that of focal contacts seen with interference reflection microscopy (IRM). These contacts had smooth external surfaces and were often arranged in chevron-shaped complexes. However, at 4–6 h, the surface texture of focal contacts became fibrous and the contact periphery was delineated with the orifices of membrane-associated vesicles. Development of this filamentous substructure is correlated with the maximum concentration of fibronectin receptors and fibronectin at focal contacts, suggesting that these molecules are involved in the maturation and stabilization of focal contacts.
Tubulin polymerization in unfertilized sea-urchin eggs induced by elevated temperature
Authors: Harris Patricia J. , Clason Elise L. , Prier Kevin R.
Published: April 2021

Spontaneous polymerization of tubulin was induced in unfertilized eggs of the sea urchins Lytechinus pictus and Strongylocentrotus purpuratus by warming to temperatures 10–1 2deg. C above the normal environment for each species. Indirect immunofluorescence microscopy showed that over a period of several hours polymerization proceeded from a fine crystal-like precipitate to larger and fewer higher-order structures. These structures differed morphologically between the two species: L. pictus formed cytasters, while S. purpuratus most often formed flame-like arrays. Nuclear cycles were not initiated, permitting longterm observation of steady-state polymer redistribution. After several hours only a few very large cytasters remained near the surface of L. pictus eggs and these tended to constrict to form protrusions or occasionally a cleavage furrow that divided the egg into two fragments. Staining with rhodamine-labeled phalloidin showed that actin was also concentrated in the cytasters, primarily at the periphery of the centrosphere-like regions. L. pictus cytasters could be recycled by repeated cooling and rewarming, and showed an accelerated rate of polymerization with each successive cycle. Cytasters could also be isolated as long as the initial procedures were carried out at the elevated temperature. Washed preparations were stable at room temperature. From these observations we conclude that: (1) species-specific cytoplasmic factors control the temperature limits for spontaneous tubulin polymerization in unfertilized eggs, as well as the form of higher-order structures that are produced; (2) tubulin polymerized in whole eggs at elevated temperature shows characteristics similar to purified tubulin io vitro, with regard to cold recycling and steady-state polymer redistribution; (3) actin also polymerizes at the elevated temperature and is found closely associated with microtubules of the cytasters.
Aggregation of membrane-associated actin filaments following localized adherence of enteropathogenic Escherichia coli to HeLa cells
Authors: Da Silva M. Lourdes Monteiro , Mortara Renato A. , Barros Helena C. , Souza Wanderley De , Trabulsi Luiz R.
Published: April 2021

We have previously observed that enteropathogenic Escherichia coli (EPEC) adhere to HeLa cells in a localized manner, which we designated localized adherence as opposed to the diffuse pattern of adhesion. In this paper we have examined the effects of localized adherence of EPEC on the actin microfilament system of host HeLa cells. Centrifugation of bacteria onto HeLa cells improved the localized adherence and rapid rearrangements of actin filaments were detected by immunofluorescence and electron microscopy. Aggregation of microfilaments is consistently observed at the sites of localized adherence, and is abolished by cytochalasin D and low temperatures. Scanning electron microscopy indicates that these aggregates are surface microvilli entangled with attached EPEC.
Definition of individual components within the cytoskeleton of Trypanosoma brucei by a library of monoclonal antibodies
Authors: Woods Angela , Sherwin Trevor , Sasse Rosemary , Macrae Thomas H. , Baines Anthony J. , Gull Keith
Published: April 2021

The detergent-insoluble T. brucei cytoskeleton consists of several morphologically distinct regions and organelles, many of which are detectable only by electron microscopy. We have produced a set of monoclonal antibodies that define each structural component of this highly ordered cytoskeleton. The monoclonal antibodies were selected by cloning of hybridomas produced from mice injected with complex mixtures of proteins of either the cytoskeleton itself or salt extracts thereof. Four antibodies define particular tubulin isotypes and locate the microtubules of the axoneme and sub-pellicular array; two antibodies recognize the flagellum attachment zone; one recognizes the paraflagellar rod and another the basal bodies. Finally, one antibody defines a detergent-insoluble component of the nucleus. The antigens detected by each monoclonal antibody have been analysed by immunofluorescence microscopy, immunogold electron microscopy and Western blotting.
Reassembly of microtubules in Nitella tasmanica: assembly of cortical microtubules in branching clusters and its relevance to steady-state microtubule assembly
Authors: Wasteneys Geoffrey O. , Williamson Richard E.
Published: April 2021

Giant internodal cells of Nitella tasmanica have cortical microtubules beneath the plasma membrane and endoplasmic microtubules associated with sub-cortical actin bundles and nuclei. We depolymerized the microtubules with oryzalin and followed their reassembly by immunofluorescence. At 18°C (the standard temperature of culture), microtubules were lost from young cells within 10 min and the first microtubules were detected in the cortex within 20 min of washing out the herbicide. Microtubules of older cells disassembled and re-formed more slowly. Continued cortical microtubule assembly was at acute angles to the first-formed microtubules, building branching clusters of microtubules. At 25°C, cortical microtubule assembly generated less extensively branched clusters and was completed more rapidly. Larger clusters but shorter MTs were generated in older cells. Reassembly of microtubules in the endoplasm only began 50 min after the removal of oryzalin. We therefore conclude that assembly proceeded independently in the cortex and endoplasm. Cortical assembly involves scattered assembly events initiating microtubules from which, as the latter elongate, further microtubules assemble as branches. We suggest that similar processes operate in steady-state cells and we explain with a simple model why branched clusters of microtubules are unusually large after microtubule depolymerization. By proposing that these processes show differential changes in activity with temperature and during cell ageing, we can account in qualitative terms for the age- and temperature-dependent differences in microtubule reassembly patterns.
Cellular asymmetry inChlamydomonas reinhardtii
Authors: Holmes Jeffrey A. , Dutcher Susan k.
Published: April 2021

Although largely bilaterally symmetric, the two sides of the unicellular alga Chlamydomonas rein-hardtii can be distinguished by the location of the single eyespot. When viewed from the anterior end, the eyespot is always closer to one flagellum than the other, and located at an angle of approximately 45° clockwise of the flagellar plane. This location correlates with the position of one of four acetylated microtubule bundles connected to the flagellar apparatus. Each basal body is attached to two of these microtubule rootlets. The rootlet that positions the eyespot is always attached to the same basal body, which is the daugher of the parental/daughter basal body pair. At mitosis, the replicated basal body pairs segregate in a precise orientation that maintains the asymmetry of the cell and results in mitotic poles that have an invariant handedness. The fusion of gametic cells during mating is also asymmetric. As a result of asymmetric, but dtfferent, locations of the plus and minus mating structures, mating preferentially results in quadriflagellate dikaryons with parallel flagellar pairs and both eyespots on the same side of the cell. This asymmetric fusion, as well as all the other asymmetries described, may be necessary for the proper photo-tactic behavior of these cells. The invariant handedness of the spindle pole, eyespot position, and mating structure position appears to be based on the inherent asymmetry of the basal body pair, providing an example of how an intracellular pattern can be determined and maintained.
Basolateral secretion of kappa light chain in the polarised epithelial cell line, Caco-2
Authors: Hughson Elaine J. , Cutler Daniel F. , Hopkins Colin R.
Published: April 2021

The immunoglobulin kappa light chain is constitutively secreted in non-polarised cells. It is therefore unlikely to display any of the signals thought to be required for the selective delivery of proteins to the apical or basolateral borders of polarised epithelial cells. We have transfected the gene for the kappa light chain into a polarised epithelial cell line (Caco-2) and shown that it is secreted predominantly from the basolateral surface. Metabolically labelled endogenous secretory products show the same polarity and we conclude, therefore, that in Caco-2 cells there is a major intracellular trafficking route to the basolateral border that requires no sorting signal.
An ultrastructural study of crossbridge arrangement in the fish skeletal muscle thick filament
Authors: Kensler Robert W. , Stewart Murray
Published: April 2021

A procedure has been developed for isolating goldfish skeletal muscle thick filaments that preserves the near-helical arrangement of the myosin crossbridges under relaxing conditions. These filaments have been examined by electron microscopy and computer image analysis. Electron micrographs of the negatively stained filaments showed a clear periodicity associated with the crossbridges, with an axial repeat every 42.9 nm. Computed Fourier transforms of the negatively stained filaments showed a series of layer lines confirming this periodicity, and were similar to the X-ray diffraction patterns of fish muscle obtained by J. Harford and J. Squire. Analysis of the computed transform data and filtered images of the isolated fish filaments demonstrated that the myosin crossbridges lie along three strands. Platinum shadowing demonstrated that the strands have a right-handed orientation, and computed transforms and filtered images of the shadowed filaments suggest that the crossbridges are perturbed both axially and azimuthally from an ideal helical arrangement.
The behaviour of microtubules in chromosomal spindle fibres irradiated singly or doubly with ultraviolet light
Authors: Wilson Paula , Forer Arthur
Published: April 2021

Areas of reduced birefringence (ARBs) produced by ultraviolet microbeam irradiation are areas of depolymerized microtubules. ARBs probably move poleward either by microtubule subunit addition at the kinetochore and loss at the pole, or by microtubule subunit addition at one edge of the ARB and loss from the other edge. In this paper we have used two approaches to try to distinguish between these two models. First, we determined whether the edges of the ARB move at the same rate; if ARB motion is due solely to addition at the kinetochore and loss at the pole, with the ARB edges unable to exchange subunits, then the two edges of each ARB should move at the same rate. On the other hand, if the exchange is at the ARB edges, then, from data from microtubules in vitro, the poleward edge should move much faster than the kinetochore-ward edge. We found that the two edges of the ARB move at the same rate about half the time, but half the time they do not. Second, we studied the behaviour of two ARBs on a single fibre. If ARB motion is due solely to subunit addition at the kinetochore and loss at the pole, then the two ARBs must move poleward together. We found that after two ARBs are formed on a single fibre the region between the ARBs is unstable and rapidly depolymerizes. These results do not fit either model and suggest that influences of kinetochores and poles or other factors need to be considered that are not duplicated in experiments on microtubules in vitro.
Book review
Authors: Cutler D.
Published: April 2021

Protein targeting is currently in a very interesting phase. For most of the pathways by which proteins traffic around the cell we now have at least preliminary descriptions obtained by morphological and fractionation techniques. Furthermore, the use of molecular biology in investigating the structure and function of the transported protein by mutagenesis and expression is making some important contributions toward our understanding. As well as these, the use of in vitro assays for transport processes has begun to complement the power of yeast genetics and, together, these are leading to the isolation of components of the machinery controlling transport.

Protein Targeting, edited by K. F. Chater, N. J. Brewin, R. Casey, K. Roberts, T. M. A. Wilson and R. B. Flavell. The Company of Biologists Ltd (obtainable from The Biochemical Society, Colchester CO2 8HP) 1989. Pp. 253. £35, $65, hardback

Protein Targeting, edited by A. P. Pugsley. Academic Press Inc. New York, 1989. Pp. xi+279. $45, hardback
A myofibrillar protein of insect muscle related to vertebrate titin connects Z band and A band: purification and molecular characterization of invertebrate mini-titin
Authors: Nave Rüdiger , Weber Klaus
Published: April 2021

We show that myofibrils of insect flight and leg muscle contain a doublet of polypeptides with apparent molecular weights of 700K (K=103Mr) (Hmp I) and 600K (Hmp H), respectively. In Locusta migratoria high ionic strength extraction solubilizes only Hmp II, which is readily purified in native form. It probably reflects a proteolytic derivative of the non-extractable Hmp I. On the basis of its viscosity radius and sedimentation coefficient, Hmp II has a molecular weight of 600K and seems to consist of a single polypeptide chain. The highly asymmetric structure of the molecule is confirmed by rotary shadowing. The flexible rods have a uniform diameter of 3-4 nm and an average length of 260 nm. Polyclonal antibodies show crossreactivity between Hmp II and its putative precursor Hmp I. We discuss the similarities and differences between the larger titin l/titin II of vertebrate sarcomeric muscle and the smaller Hmp l/Hmp II of invertebrate muscle and conclude that the latter may reflect a mini-titin. In line with the smaller length, immunoelectron microscopy locates the insect mini-titin to the I band and a very short portion of the A band only, while vertebrate titin is known to connect the Z band to the M band. Mini-titin has also been purified from several other insects including Drosophila. Immunofluorescence microscopy on frozen sections shows that mini-titin is present in the sarcomeric muscles of various species from different invertebrate phyla. These include Annelida, Nematomorpha, Plathelmintha, Nemertea and Nematoda like Ascaris lumbricoides and Caenorhabditis elegans. This wide-spread occurrence of invertebrate mini-titin is confirmed by immunoblotting experiments.
Changes in cyclic AMP receptor properties during adaptation in Dictyostelium discoideum
Authors: Ludérus M. E. E. , Spijkers M. J. , Driel R. Van
Published: April 2021

In developing Dictyostelium discoideum cells, binding of cyclic AMP to the chemotactic receptor has been shown to oscillate. These oscillations represent cycles of activation, adaptation and deadaptation of the cyclic AMP receptor system.

We studied the molecular basis of these oscillatory changes in cyclic AMP receptor binding. We developed a rapid method of lysing cells during the course of the oscillations. This method guaranteed good preservation of ligand binding properties of the cyclic AMP receptor.

We found that oscillations in cyclic AMP binding resulted from changes in receptor affinity. The total number of receptors did not significantly change during oscillations. Our experiments also showed that both GTP and GDP abolished oscillations in receptor binding completely, presumably by acting via a G protein. The guanine nucleotides reduced the affinity of the receptor at all time-points of the oscillation cycle to the minimal, i.e. adapted, level.

We conclude that the cyclic process of activation, adaptation and de-adaptation in D. discoideum, at cyclic AMP receptor level, involves changes in receptor-G protein interaction. During adaptation, the affinity of the cyclic AMP receptor decreases and the receptor becomes insensitive to guanine nucleotides.
High-resolution surface views of human lymphocytes during capping of CD4 and HLA antigens as revealed by immunogold fracture-flip
Authors: Pavan Antonio , Mancini Patrizia , Lucania Giuseppe , Frati Luigi , Torrisi Maria Rosaria , Silva Pedro Pinto da
Published: April 2021

The surface ultrastructure of lymphocytes during capping of two transmembrane proteins is shown. As seen by fracture-flip the plasma membranes of human lymphocytes are covered by a high density of surface particles. Incubation in 30 % glycerol leads to aggregation of these surface particles. Immunogold labelling shows that the transmembrane proteins bearing HLA class I and CD4 antigens are confined to the particle aggregates. These results indicate that surface particles revealed by fracture-flip represent surface protrusions of integral membrane proteins seen as intramembrane particles in freeze-fractured lymphocytes.

During capping HLA or CD4 antigens aggregate into progressively larger patches and, finally, into single caps. As revealed by fracture-flip the patches/caps are seen as clearly differentiated raised platforms that are clearly and sharply demarcated relative to contiguous areas of the surface. In nonpatched (non-capped) regions, the pattern of distribution and apparent density of surface particles remain unaltered. Immunogold labelling clearly demarcates patches and caps, and shows that virtually no antigen molecules remain dispersed over the non-patched (non-capped) regions. Estimates of the surface density of either HI.A or CD4 antigens over the capped areas point to high planar concentrations of the transmembrane proteins that bear these antigens.
The protein kinase c inhibitor H-7 activates human neutrophils: effect on shape, actin polymerization, fluid pinocytosis and locomotion
Authors: Keller H. U. , Niggli V. , Zimmermann A. , Portmann R.
Published: April 2021

The present study demonstrates new properties of H-7. The protein kinase inhibitor H-7 is a potent activator of several neutrophil functions. Stimulation of initially spherical nonmotile neutrophils elicits vigorous shape changes within a few seconds, increases in cytoskeletal actin, altered F-actin distribution, increased adhesiveness and a relatively small increase in pinocytic activity. H-7 has also chemokinetic activities. Depending on the experimental condition, H-7 may elicit or inhibit neutrophil locomotion. It failed to induce chemotaxis. Thus, the response pattern elicited by H-7 is different from that of other leukocyte activators such as chemotactic peptides, PMA or diacylglycerols. The finding that H-7 can elicit shape changes, actin polymerization and pinocytosis suggests that these events can occur without activation of protein kinase C (PKC). PMA-induced shape changes and stimulation of pinocytosis were not inhibited by H-7.
Regulation by calcium of proliferation and morphology of normal human tracheobronchial epithelial cell cultures
Authors: Chopra D. P. , Sullivan J. K. , Reece-Kooyer S.
Published: April 2021

Human tracheobronchial epithelial cells have been serially passaged in serum-free medium. This serumfree model was employed to investigate the effects of different concentrations of Ca2+ (0.1, 1.0 and 2.0 mM) on multiplication and morphology of the cells. The responses were analysed in terms of growth kinetics, histochemical and ultrastructural alterations. Culturing of the cells in high Ca2+ (1.0-2.0mM) medium stimulated cell multiplication characterized by increased colony forming efficiency, greater number of cells per colony and cell population doublings per day. Additionally, the high Ca2+ concentrations induced proliferation in cultures grown to confluency in low Ca2+, (0.1 mM) medium. Cells propagated in low Ca2+ medium consisted of relatively heterogeneous cell populations, with mosteells staining positive with periodic acid-Schiff (PAS) reagent. Ultrastructurally the cells exhibited secretory vesicles and microvilli on their surfaces, small desmosomes and intercellular interdigitation between cells and numerous large secretory vesicles in the cytoplasm. The cells grown in high Ca2+ medium acquired characteristics of a highly proliferative phenotype. The cultures consisted of closely packed, relatively homogeneous cells that did not stain with PAS reagent. Their characteristic features were: absence of surface secretory vesicles, reductions of microvilli and intercellular interdigitations, and increases in size and number of desmosomal junctions. The results show that low Ca2+ in the culture medium inhibits cell multiplication and favors the secretory cell phenotype, while high Ca2+ levels stimulate cell multiplication and inhibit the secretory cell phenotype.
Monensin does not consistently inhibit the spreading of normal or duchenne fibroblasts on glass
Authors: Brown Susan C. , Fisher Derek , Lucy Jack A.
Published: April 2021

The spreading on glass of monensin-treated normal and Duchenne fibroblasts has been investigated with the intention of extending this approach to a study of the comparative spreading of these cells on differing substrata. Untreated normal and Duchenne fibroblasts varied considerably in their ability to spread on glass. The spreading properties of normal and DMD fibroblasts treated in four different ways were compared: (1) pre-incubated and plated without monensin; (2) pre-incubated with, but plated without monensin; (3) pre-incubated without, but plated with monensin; (4) pre-incubated and plated with monensin. The response to plating with monensin (and preincubation/plating with monensin) also varied from patient to patient, but no statistically significant differences in the degree of spreading between the four treatment groups were observed in pooled data for either normal or dystrophic fibroblasts. Our data thus do not substantiate the previous finding of Pizzey et al. (1984) that Duchenne fibroblasts spread less well than normal fibroblasts after pre-incubation or plating with monensin, and possible explanations for this are discussed. The observations made are, however, consistent with the recent report that dystrophin is effectively not expressed in fibroblasts, and with the idea that the abnormal behaviour of endomysial fibroblasts in Duchenne dystrophy is a secondary consequence of their proximity to degenerating muscle.
Endocytosis in elongating root cells of Lobelia Erinus
Authors: Samuels A. L. , Bisalputra T.
Published: April 2021

Endocytosis was demonstrated in elongating cortical and epidermal root cells of Lobelia erinus using the apoplast marker lanthanum nitrate. Lanthanum treatment produced electron-dense deposits throughout the cell wall, as well as in coated and smooth vesicles, partially coated reticula, and multi-vesicular bodies. This labelling pattern was observed in root cells that had been ultrarapidly frozen on a copper mirror and freeze-substituted (cryofixation) or fixed by conventional transmission electron microscope (TEM) techniques. The amount of endocytosis occurring was measured by counting the number of vesicles μm -2 in root cells at various stages of development. Endocytosis occurred most in actively elongating cells, and least in mature cells, which were no longer elongating. The relationship between endocytosis and active cell wall secretion suggests that endocytosis may be acting to remove excess plasma membrane material added during exocytosis of secretory vesicles.
Identification of proteins associated with microtubule-organizing centres and filaments in the oral apparatus of the ciliate Paramecium tetraurelia
Authors: Keryer Guy , Iftode Francine , Bornens Michel
Published: April 2021

In an effort to study the assembly of microtubules in ciliates we have isolated the oral apparatus of Paramecium tetraurelia, a major microtubule-organizing centre (MTOC) and identified several proteins localized in the fibrillar system associated with basal bodies. Using a monoclonal antibody raised against human centrosomes (CTR210) and a polyclonal antibody (OF1) directed against a 87×10-3Mr protein of the oral apparatus of Tetrahymena, we observed the same decoration of the oral apparatus of Paramecium, both in situ and after isolation. Ultrastructural localization further showed the presence of the antigens in the fibrillar network that forms a layer under almost all the buccal cavity in close apposition to the basal bodies. CTR210 recognized two sets of polypeptides of Mr72 and 80×103, whereas OF1 recognized a single polypeptide of 80×103. Only the 80×103Mr polypeptide was also decorated with the monoclonal antibody MPM-2, previously shown to decorate the oral apparatus of Paramecium and known to react with phosphorylated epitopes in a large variety of MTOCs. All the proteins identified with the three antibodies are insoluble at high ionic strength, display several isoforms and apparently belong to the same fibrillar material. The function of this material in the spatial organization, the structure, and activity of the MTOCs is discussed.
Segregation of the myogenic cell lineage in mouse muscle development
Authors: Jones Gareth E. , Murphy Susan J. , Watt Diana J.
Published: April 2021

With increasing interest in the idea of therapeutic implantation of normal muscle precursor cells into muscle lacking the protein product of the dystrophin gene, it has become important to obtain enriched populations of myogenic cells from biopsied muscle sources. Myogenic cells for implantation are highly favoured as they are the only cells that will fuse readily with host muscle fibres into which they are implanted, thus carrying the introduced gene into the target fibre with the maximum of efficiency. Second, myogenic cells appear less immunogenic than those of a non-myogenic nature; and third, the use of mononuclear myogenic cells may permit the introduction of multiple copies of a deficient gene into the patient’s own cells.

From a mixed population of cells obtained by the enzymic disaggregation of neonatal murine muscle we have selected, utilising a modification of the panning technique, for a cell population rich in myogenic cells. Segregation was accomplished using Mab H28, an antibody to the mouse neuronal cell adhesion molecule (N-CAM), derived from mouse/rat hybridoma cells. Following incubation with Mab H28, disaggregated muscle was applied to the surface of a bacteriological grade dish previously coated with anti-rat immunoglobulin. Cells segregated into two populations; those bearing N-CAM, and hence labelled with Mab H28, were adherent to the dish, whereas those not expressing N-CAM remained in suspension.

Use of this technique, which involves minimal cell loss, resulted in the segregation of prefusion myogenic cells together with fibroblasts in the ‘nonadherent’ fraction, whereas cells in the adherent fraction consisted of a highly enriched population of actively dividing myogenic cells.
Proteins of the mammalian mitotic spindle: phosphorylation/dephosphorylation of MAP-4 during mitosis
Authors: Vandré Dale D. , Centonze Victoria E. , Peloquin John , Tombes Robert M. , Borisy Gary G.
Published: April 2021

The phosphoprotein composition of isolated CHO spindles was analyzed using the MPM-1 and MPM-2 antibodies, which are reactive with a phosphorylated epitope enriched in mitotic cells and present on the centrosome, kinetochores, midbody and fibers of the mitotic spindle. Several high molecular weight phosphorylated spindle proteins were detected on immunoblots, including species of 410 ×103Mr, 350 ×103Mr, a 230–240×103Mr doublet, 210×103 Mr and 120×103Mr. The temporal and spatial distribution of the MPM-reactive phosphoproteins was determined by examining spindle structures isolated from cells at various stages of mitosis. The susceptibility of the staining pattern to extraction with salt, a procedure known to remove most microtubule-associated proteins (MAPs), was also examined.

The phosphorylated 210×103Mr species was identified as MAP-4 and localized to the spindle fibers using (1) a polyclonal antibody raised against this species, that reacted with known MAPs, and (2) established MAP-4 antibodies that reacted with the spindle 210 × 103Mr MPM-reactive proteins. The comparative immunoblot and immunofluorescence analysis establishes a cycle of phosphorylation/ dephosphorylation of MAP-4 upon entry and exit from mitosis. Regarding the other MPM-reactive proteins, comparative immunofluorescence staining and immunoblot analysis of isolated spindle samples before and after salt extraction indicate that they may be constituents of the centrosome, kinetochores or midbody, but their definitive identification awaits the production of monospecific antibodies..
Immunoelectron microscopic analysis of the intracellular distribution of primer recognition proteins, annexin 2 and phosphoglycerate kinase, in normal and transformed cells
Authors: Kumble Krishnanand D. , Vishwanatha Jamboor K.
Published: April 2021

Primer recognition proteins (PRP) are accessory proteins for DNA polymerase a in lagging strand DNA replication. We have previously reported that the PRP consist of a complex of two proteins identified as 3-phosphoglycerate kinase (PGK) and the protein-tyrosine kinase substrate, annexin 2 monomer. The physiological role of annexin 2 is not known. Two pools of annexin 2 exist in cells. A majority of annexin 2 is localized with the plasma membrane as a heterotetramer in association with a light chain. Monomer annexin 2 is cytosolic. The identification of annexin 2 monomer as a part of the PRP complex represents one of the physiological roles of this protein in cells. To function as PRP, annexin 2 and PGK would have to be present in the cell nucleus. To investigate whether monomer annexin 2 is indeed associated with nuclear DNA synthesis, we investigated the presence of annexin 2 and PGK in the cell nucleus. In this paper, we demonstrate the presence of annexin 2 and PGK in nuclear extracts. The nuclear fraction of these proteins represents a small subset of the total cellular pools. Immunoelectron-microscopic analyses using anti-PRP antisera demonstrate the distribution of these proteins in HeLa cell nuclei and cytoplasm. Under identical conditions, an anti-cyto-keratin monoclonal antibody preferentially labels the plasma membrane without detectable intracellular staining. The distribution of annexin 2 and PGK in both nuclei and cytoplasm is similarly observed in cells from normal tissues such as freshly isolated rat hepatocytes and hamster pancreatic tissue. The results presented in this report further substantiate the involvement of annexin 2 and PGK, as part of the PRP complex, in nuclear DNA synthesis.