bluuebunny/crossref_metadata_2025 huggingface dataset:
Rectilinear particle arrays in freeze-fracture replicas of the surface membrane of Paramecium tetraurelia
Authors: Preston Robin R. , Newman T. M.
Published: April 2021

Freeze-fracture replicas of the plasma membrane of unfixed, uncryoprotected Paramecium tetraurelia bear large rectilinear arrays of 11 nm particles arranged in 7–11 parallel rows. The arrays are of sufficient size to leave impressions in replicas of the underlying outer alveolar membrane, and are apparent as parallel ridges in replicas of the surface coat of deep-etched cells.

By noting the location of arrays in replicas of identified portions of the cortex of P. tetraurelia, it has been possible to map the distribution of arrays over the cell surface. The arrays are found primarily over the anterior surfaces of the cell, covering an area that extends from the preoral suture over the left adoral field and a large portion of the anterior dorsal surface.

Freeze-fracture analyses of cells taken from a number of different stages of a culture cycle suggest that the particle arrays are not replicated as an integral part of the cortex during cell division, but are assembled and oriented in the membrane as the cells mature. The appearance of small intramembranous particle complexes in the plasma membrane of cells in logarithmic growth phase supports this hypothesis, possibly representing an assembly stage in the formation of the larger particle arrays.

The facts that the particle arrays are apparent in replicas of the surface coat of cells, are found primarily at the anterior of the cell body, and have a highly specific orientation with respect to the cell surface, strongly suggest that they function as chemoreceptors in P. tetraurelia.
Chemotactic peptide-induced exocytosis in neutrophils: granule fusion patterns depend on the source of messenger calcium
Authors: Chandler Douglas E. , Kazilek Charles J.
Published: April 2021

Rabbit neutrophils, exposed either to partially purified human C5a or to formylmethionyl-leucyl-phenylalanine in the presence of 5μgml-1 cytochalasin B underwent a rapid, compound exocytosis. The pattern of granule fusion, as visualized in freeze-fracture replicas, differed depending on the source of messenger calcium. In the presence of extracellular calcium, a linearly directed pattern, consisting of finger-like invaginations converging at the cell centre, was prominent at 10–20 s after stimulation. After stimulation for 20–40 s, further fusion of granule membranes created extremely convoluted surfaces, some consisting of up to a dozen granule membranes connected by narrow pores or flat ribbons of membrane. In many cells the peripheral cytoplasm was constricted to form lobes or finger-like protrusions. Neutrophils stimulated in the absence of extracellular calcium exhibited granule fusion in the directed pattern, with only occasional involvement of the convoluted patterns seen when calcium is present. In contrast, neutrophils depleted of intracellular calcium before stimulation (and thereby forced to use extracellular calcium for triggering secretion) exhibited the convoluted pattern of granule fusion almost exclusively. These results suggest that the directed pattern of fusion is initiated by release of intracellular calcium or a calcium-independent pathway and that the non-directed, convoluted pattern of fusion is initiated by entry of extracellular calcium.
Elemental content of mast cell granules measured by x-ray microanalysis of rat thymic tissue sections
Authors: Kendall Marion D. , Warley Alice
Published: April 2021

Mast cell granules were examined by fully quantitative X-ray microanalysis of 20 cells in freeze-dried cryosections. The mast cells were situated mainly in the connective tissue of the thymic capsule of five adult male Carworth Sprague Europe rats. In addition 30 red blood cells were analysed from the same sections.

Nineteen of the mast cells had granules rich in S and K. One cell had smaller granules, and in this cell the granules contained high [Ca] and [P] instead of high [S] and [K], In the majority of cells (13) the S: K ratio was highly correlated and less than 2–2, whereas in the remaining six cells the individual granule ratios were very variable in any one cell and much higher. The mean granule [K] (994 ± 57 mmol kg-1 dry wt) was about four times the mean cytoplasmic level of 227 ±81 mmol kg-1 dry wt. The existence of this difference in concentration between the granules and the cytoplasm suggests that the K in the granules must be bound.

The relationship between the [K] and [S] is discussed with regard to the possible binding of heparin and amines in the granules.
Chromosome condensation activity in ovulated metaphase ii mouse oocytes assayed by fusion with interphase blastomeres
Authors: Czołowska Renata , Waksmundzka Małgorzata , Kubiak Jacek Z. , Tarkowski Andrzej K.
Published: April 2021

Fusion of large and small karyoplasts produced from metaphase II mouse oocytes with interphase blastomeres from 2-cell and 8-cell embryos (volume ratio of partners, 1:1) results in premature chromosome condensation (PCC) of the interphase nucleus in the majority of the fusion products (hybrids). Fused under the same experimental protocol, oocyte-derived cytoplasts also induce PCC of the blastomere nucleus in the fusion products (cybrids) provided they originate from recently ovulated oocytes (14½ −15h after injection of human chorionic gonadotrophin (HCG)). In cytoplasts derived from older oocytes (16–20 h post-HCG) chromosome condensation activity gradually decreases with time as can be inferred from the increasing proportion of cybrids retaining interphase blastomere nuclei. However, even the oldest cytoplasts (19–20 h post-HCG) can induce PCC if the cytoplast volume significantly exceeds the volume of the interphase partner (7:1)-

We postulate that the condensation activity is predominantly bound to the nuclear apparatus (most probably to the chromosomes), and that in the cytoplasm of metaphase II mouse oocyte it decreases with post-ovulatory age.
Neutrophil leucocyte chemotaxis is not induced by a spatial gradient of chemoattractant
Authors: Vicker Michael G. , Lackie John M. , Schill Walter
Published: April 2021

Chemotaxis and directed locomotion of neutrophil leucocytes are generally thought to be determined by the directed response of the cell to stable, spatial gradients of chemoattractants. In most cases, however, cells are also exposed to characteristic temporal changes in the attractant concentration during the lifetime of the gradient, especially as it develops. We have attempted to test whether neutrophils can respond to a spatial gradient in which these temporal changes are essentially absent. Gradients of formyl-peptides were made across a narrow barrier of agarose gel that separated two fluid reservoirs, and the cells were observed cinematographically as they moved between gel and glass. In gradients predeveloped at low temperature, at which cell motion and responses to attractant were inhibited, neutrophils showed no tendency to accumulate up-gradient when warmed to 37°C. Yet their speed and turning behaviour was related to the local concentration of formyl-peptide. However, gradients that developed at 37°C, whilst the cells were responsive, elicited directed locomotion. We also tested populations that were either spreading into or already evenly distributed across micropore filters to see how cells might sense directional cues. We reasoned that evenly distributed populations could accumulate in a spatial gradient only if cells were able to ‘read’ it. However, no redistribution occurred without an applied impulse of attractant. It seems that the oriented, temporal component of an attractant signal is essential if a directed response (i.e. non-random turning) is to occur; a spatial gradient of soluble attractant alone does not induce neutrophil accumulation or taxis. This finding has implications for the termination of the acute inflammatory response, for clinical tests of leucocyte behaviour and for morphogen signal interpretation by cells in developing tissues.
Actin during pollen germination
Authors: Heslop-Harrison J. , Heslop-Harrison Y. , Cresti M. , Tiezzi A. , Ciampolini F.
Published: April 2021

The cytoplasm of the vegetative cell of the ungerminated pollen grain of Endymion non-scriptus and other angiosperm species contains numerous fusiform bodies sometimes exceeding 15 gm in length and 2-5 gm in width, which bind fluorescent-labelled phalloidin and are likely therefore to constitute a storage form of actin. The bodies are dispersed during the activation of the pollen, being replaced by aggregates of slender phalloidin-binding fibrils, which converge towards the germination apertures and are present in the emerging pollen tube. The storage bodies appear to be homologous with crystalline-fibrillar structures, shown in an earlier paper to be abundantly present in the vegetative cells of Nicotiana pollen. These are composed of massive aggregates of linearly disposed units with individual widths of 4–7 nm, probably to be interpreted as actin microfilaments.

Vegetative-cell protoplasts from mature but ungerminated pollen disrupted in osmotically balancing medium release extended phalloidin-binding fibrils of a kind not observed in the intact grain. It is suggested that these are derived by the rapid dissociation of the compact actin storage bodies present in the vegetative cell at this stage of development.
Presumptive mesoderm cells from Xenopus laevis gastrulae attach to and migrate on substrata coated with fibronectin or laminin
Authors: Nakatsuji Norio
Published: April 2021

During amphibian gastrulation, presumptive mesoderm cells migrate from the blastopore towards the animal pole along the inner surface of the ectodermal layer. Their natural substratum is a network of anastomosing extracellular matrix fibrils, which contains fibronectin and laminin, as shown by immunostaining. If the fibril network is transferred onto a coverslip from the ectodermal layer, dissociated mesodermal cells readily attach to such conditioned surfaces and show active migration in a medium of high pH and low calcium ion concentration.

In the present study, the surface of tissue culture dishes was coated with fibronectin, laminin, collagen type IV or heparan sulphate, to examine the effects on cell attachment and movement. The presumptive mesoderm cells from Xenopus laevis gastrulae showed rapid adhesion and active movement on the fibronectin- or laminin-coated surfaces. Cell adhesion was stronger and the mean rate of movement was higher on the fibronectin-coated surface than on the laminin-coated surface. The dissociated ectodermal cells did not attach to the fibronectin- or laminin-coated surfaces. The mesodermal cells did not attach to the collagen-, or heparan sulphate-coated surfaces, showing that these components of the basement membrane cannot serve as an adequate substratum for the mesoderm cells, at least by themselves.
Invasive behaviour of mouse primordial germ cells in vitro
Authors: Stott David , Wylie Christopher C.
Published: April 2021

We have isolated migrating primordial germ cells (PGCs) from 10-5-day mouse embryos and studied their behaviour when cultured on a mouse embryo fibroblast (STO) cell line. Living and fixed PGCs were identified by fluorescent labelling with a monoclonal antibody specific for PGCs in the culture system used. The behaviour of the cells was studied using interference reflexion microscopy (IRM) and time-lapse video cinematography. The IRM pattern displayed by PGCs is typical of highly motile cell types, the cells lack focal contacts and possess large areas of close contacts indicative of weak membrane to substrate interaction. The PGCs exhibit relatively high rates of translocation and lack contact inhibition. They were observed to underlap STO cells in subconfluent monolayers and to penetrate between the cells of confluent monolayers, becoming located between the monolayer and its substrate. These observations support the hypothesis that migrating mouse PGCs are inherently motile and are able transiently to disrupt the adhesion of surrounding cells. These results suggest that PGCs actively migrate to the developing gonad in vivo.
Algal endosymbiosis in brown hydra: Host/symbiont specificity
Authors: Rahat M. , Reich V.
Published: April 2021

Host/symbiont specificity has been investigated in non-symbiotic and aposymbiotic brown and green hydra infected with various free-living and symbiotic species and strains of Chlorella and Chlorococcum. Morphology and ultrastructure of the symbioses obtained have been compared. Aposymbiotic Swiss Hydra viridis and Japanese H. magnipapillala served as controls.

In two strains of H. attenuata stable hereditary symbioses were obtained with Chlorococcum isolated from H. magnipapillata. In one strain of H. vulgaris, in H. oligactis and in aposymbiotic H. vtrtdis chlorococci persisted for more than a week. Eight species of free-living Chlorococcum, 10 symbiotic and 10 free-living strains of Chlorella disappeared from the brown hydra within 1-2 days.

In H. magnipapillata there was a graded distribution of chlorococci along the polyps. In hypostomal cells there were >30 algae/cell while in endodermal cells of the mid-section or peduncle <10 algae/cell were found. In H. attenuata the algal distribution was irregular, there were up to five chlorocci/cell, and up to 20 cells/hydra hosted algae.

In the dark most cells of Chlorococcum disappeared from H. magnipapillata and aposymbiotic hydra were obtained. Chlorococcum is thus an obligate phototroph, and host-dependent heterotrophy is not required for the preservation of a symbiosis. The few chlorococci that survived in the dark seem to belong to a less-demanding physiological strain.

In variance with known Chlorella/H. viridis endosymbioses the chlorococci in H. magnipapillata and H. attenuata were tightly enveloped in the vacuolar membrane of the hosting cells with no visible perialgal space. Chlorococcum reproduced in these vacuoles and up to eight daughter cells were found within the same vacuole.

We suggest that the graded or scant distribution of chlorococci in the various brown hydra, their inability to live in H. viridis and the inability of the various chlorellae to live in brown hydra are the result of differences in nutrients available to the algae in the respective hosting cells.

We conclude that host/symbiont specificity and the various forms of interrelations we show in green and brown hydra with chlorococci and chlorellae are based on nutritional-ecological factors. These interrelations demonstrate successive stages in the evolution of stable obligatoric symbioses from chance encounters of preadapted potential cosymbionts.
Cytoplasmic organization during pattern formation in the alga Micrasterias
Authors: Mcnally James G. , Swift Hewson
Published: April 2021

Using an improved fixation for electron microscopy, we have observed an ordered cytoplasmic organization during pattern formation in the alga Micrasterias. At the earliest stages of morphogenesis (the bulge to 3-lobe stage), as observed previously, electron-dense vesicles and endoplasmic reticulum were found towards the cell periphery, while mitochondria and Golgi material were located internally. In contrast to previous studies, which found no organization in the cytoplasm of growing lobes, we observed a single cluster of electron-dense vesicles at the tip of each lobe at or beyond the 5-lobe stage. In lobes about to branch, two separate clusters of electron-dense vesicles were present in each lobe, apparently foreshadowing the sites of new lobe outgrowth. Individual lobes also possessed an ordered distribution of organelles. Immediately basal to the lobe tip, the concentration of electron-dense vesicles decreased and rough endoplasmic reticulum was more prevalent, followed more deeply in the cytoplasm by increased concentrations of mitochondria and then Golgi material. This cytoplasmic organization in individual lobes is characteristic of tip growth, thus suggesting that individual lobes elongate by this process. We have also investigated the effect of the ionophore A23187 on this cytoplasmic organization. The polarization of rough endoplasmic reticulum, mitochondria and Golgi material was destroyed by ionophore treatment in the presence of 2mM-MgCl2 or 2mM-CaCl2. The tip-associated clusters of electron-dense vesicles were dispersed by A23187 in 2mM-CaCl2 but not significantly affected by A23187 in 2mM-MgCl2. The latter observation indicates that the vesicle pattern can be disrupted by high calcium, but not high magnesium concentrations.
Characterization of a spontaneously transformed pulmonary embryonic rat (PER) epithelial cell line
Authors: Paye M. , Etievant CH. , Michiels F. , Pierard D. , Nusgens B. , Lapière CH. M.
Published: April 2021

A spontaneously transformed pulmonary embryonic rat epithelial cell line (PER) is described in terms of growth, tumorigenicity, growth factor responsiveness and biosynthetic capacity. At low-passage subcultures, PER cells grew as a monolayer and did not form colonies in soft agar. After long-term subcultivation, they lost contact inhibition, became anchorage-independent and formed tumours in nude mice. Low concentrations of foetal calf serum permit the maximum growth rate. The multiplication and metabolic activity, assessed by 2-deoxy-D-glucose uptake, was significantly stimulated by growth factors. PER cells synthesized collagen types I, III, IV and V, laminin and fibronectin, and organized a pericellular matrix made up of only basement membrane components (type IV collagen and laminin) and fibronectin. These data enabled us to define PER cells as a transformed epithelial cell line evolving towards malignancy with long-term subcultivation. These cells appeared to be a valuable tool in studies of tumour cell-matrix interactions and regulation of growth factor receptors in tumorigenesis.
Commentary cytoplasmic transport in axons
Authors: Amos L. A. , Amos W. B.
Published: April 2021

Transport along axons and dendrites ranges from the fast movement of vesicles (50–500 mm day−1), which can be observed directly by light microscopy, to the slow progress of cytoskeletal proteins. The latter, which has been followed only by using radiolabelled proteins (reviewed by Lasek et al. 1985), occurs at 0·2–8·0 mm day−1, but this is still faster than diffusion. The differential speeds of various components exclude cytoplasmic streaming models; instead, it is thought that transport is effected by molecular motors generating force between specific components of the axoplasm. Recent work has illuminated several aspects of these processes, with implications for transport in less specialized cells.
Kinesin from pig brain studied by electron microscopy
Authors: Amos L. A.
Published: April 2021

Kinesin, a protein that transports particles along microtubules, has been purified from pig brain, following published methods. By electron microscopy of both shadowed and negatively stained specimens, it appears to be a rod with a large branched structure at one end and a small fork at the other. The total length of the structure is about 100 nm. The rod has a diameter of 2–4 nm. Close to the middle of the rod there may be a flexible joint. The molecules appear to be attached to microtubules most often by their forked ends in the presence of AMP · PNP. However, the large branched end can also attach to microtubules and individual molecules occasionally have been seen cross-linking two microtubules.
Further characterization of the defects of skin fibroblasts from cancer patients
Authors: Azzarone B. , Macieira-Coelho A.
Published: April 2021

We have previously shown that skin fibroblasts from breast cancer patients display abnormal growth properties when compared with cells from patients with benign breast lesions. In the present study, we shown that it is possible to define, within the patients previously analysed, a subgroup whose fibroblasts exhibit a significant fraction of cells still synthesizing DNA when growth curves reach a plateau. The phenomenon can also be detected in skin fibroblasts from patients with other types of cancer. In two instances detection of this defect preceded the discovery of the disease.

The high percentage of labelled interphases when cell counts reach a plateau is not due to an increased duration of S phase, relative to total cell cycle duration. The data suggest that these cells are delayed in the G2 phase.

This assay buttresses our previous results, which suggested that at least in some instances cancer is a systemic disease; it could be used for the screening of patients at high risk of cancer.

Research on the patients’ somatic cells could shed more light on the process leading to neoplasia, rather than the study of the tumour cells that have already gone through several steps of the evolution to malignancy.
A simple microcomputer-based system for real-time analysis of cell behaviour
Authors: Dow Julian A. T. , Lackje John M. , Crocket Kenneth V.
Published: April 2021

An image analysis package based on a BBC microcomputer has been developed, which can simultaneously track many moving cells in vitro. Cells (rabbit neutrophil leucocytes, BHK C13 fibroblasts, or PC12 phaeochromocytoma cells) are viewed under phase optics with a monochrome TV camera, and the signal digitized. Successive frames are acquired by the computer as a 640 × 256 pixel array. Under controlled lighting conditions, cells can readily be isolated from the background by binary filtering. In real-time tracking, the positions of a given cell in successive frames are obtained by searching the area around the cell’s centroid in the previous frame. A simple box-search algorithm is described, which proves highly successful at low cell densities. The resilience of different search algorithms to various exceptional conditions (such as collisions) is discussed. The success of this system in real-time tracking is largely dependent upon the leisurely speed of movement of cells, and on obtaining a clean, high quality optical image to analyse. The limitations of this technique for different cell types, and the possible configurations of more sophisticated hardware, are outlined. This system provides a versatile and automated solution to the problem of studying the movement of tissue cells.
Induced stabilization of the transmembrane potential of Drosophila cells by heat shock and periodic applications of chloramphenicol
Authors: Jürgen Behnel H. , Weckbart G.
Published: April 2021

The transmembrane potential of Drosophila salivary gland cells is largely decreased (by 78% within 120 min) in response to the application of 5 mM-chloramphenicol (CAP), with an initial slope of 0 ·5 mV min−1. This depolarization is reversed immediately after the CAP concentration is reduced to 0 ·06 mM by step-wise dilution with normal medium. At the same concentration, thiamphenicol (TAP) induces only a small reversible depolarization by less than 30% within 120 min. These results are in agreement with the different effects of CAP and TAP on respiration and induction of the heat-shock genes, as known from previous data.

The extent of induced membrane depolarization decreases with the number of repeated applications of CAP to the same cell, alternating with 75-min periods of recovery. Moreover, reduced sensitivity to CAP is also observed in cells recovering from a transient heat shock (30 min, 36°C) 45 min prior to the addition of CAP. This phenomenon is inhibited by cycloheximide (0 ·lmM), which suggests an involvement of heat-induced proteins in the stabilization of certain membrane functions.
Structure and function of the nematosome
Authors: Ockleford C. D. , Nevard C. H. F. , Indans I. , Jones C. J. P.
Published: April 2021

The ultrastructural morphology of human placental and mouse placental nematosomes has been investigated. The description includes a three-dimensional analysis of the shape of the organelles based on serial sectioning, measurements of the repeat distance of the subunit fibre of the organelle derived by optical diffraction analysis and the results of an ultrastructural cytochemical study designed to test whether the organelle contains nucleic acid.
The distribution of a ‘mitosis-specific’ antigen during Drosophila development
Authors: Millar Sarah E. , Freeman Matthew , Glover David M.
Published: April 2021

We have used MPM-2, a monoclonal antibody raised against mitotic HeLa cells, to stain a Drosophila cell line, whole mounts of Drosophila embryos, and sectioned tissue from embryonic and larval stages of development. MPM-2 recognizes a major phosphoprotein of approximately 125 ×103Mr in Drosophila tissue culture cells that, like the mammalian MPM-2 antigen, appears to be recognized only in mitotic cells. During early embryogenesis, when the embryonic nuclei divide as a syncytium with a very short nuclear division time, MPM-2 antigen is observed within the spindle compartment at all stages of the nuclear division cycle. Upon cellularization of the embryo and lengthening of the duration of the cycle, the antigen is predominantly seen in mitotic cells. Drosophila larvae contain both diploid and polytene tissues: in diploid tissue MPM-2 staining is specifically observed over mitotic cells, as expected from its distribution in cellu-larized embryos. Surprisingly, antigen is also detected in the nuclei of polytene cells that replicate their DNA but do not undergo mitosis.
Cyclic amp stimulates accumulation of inositol trisphosphate in Dictyostelium
Authors: Europe-Finner G. Nicholas , Newell Peter C.
Published: April 2021

Previous studies indicated that in Dictyostelium amoebae signal transmission from cell surface cyclic AMP receptors to intracellular events concerned with chemotaxis involves inositol 1,4,5-trisphosphate (1,4,5-IP3): micromolar amounts of 1,4,5-IP3 or Ca2+ were found to mimic the effects of chemoattractants and 1,4,5-IP3 triggered release of Ca2+ from non-mitochondrial stores. Here we report a more direct test of the involvement of inositol phosphates. Intact amoebae were labelled with high specific activity [3H]inositol, then stimulated with the chemoattractant cyclic AMP at 22 °C and rapidly assayed for phosphorylated inositol products formed. Labelled IP3 was found to accumulate transiently after a pulse of 50nM-cyclic AMP, with a peak at 15 s after stimulation and some (inconclusive) evidence for a more rapidly formed peak at 5 8 or less. Inositol bisphosphate (IP2) showed a transient shallow peak at about 20 s. When the events of signal transmission were slowed down by incubation at 4°C, the rapidly formed IP3 peak could be consistently seen at 5 s after stimulation and the second peak at 25–30 s. Further resolution of the IP3 peaks indicated the presence of IP4, which represented a major fraction of the peak accumulated at 5 s (4°C). The results provide an important link in the chain of evidence connecting the cell surface cyclic AMP receptors, vía IP3, with the Ca2+-activated events of chemotaxis.
Extracellular matrix and cell migration: locomotory characteristics of mos-11 cells within a three-dimensional hydrated collagen lattice
Authors: Noble P. B.
Published: April 2021

The locomotory trajectories of MOS-11 cells migrating in a three-dimensional hydrated collagen lattice have been determined using a computer-assisted optical sectioning unit. The trajectories have been quantified using a three-dimensional continuous-time Markov probability theory consisting of eight directional states and one stationary state; in the latter the cells are not locomoting. Markov analysis shows that these cells are locomoting in a random manner with regard to direction and remain stationary for about three times as long as they are locomoting. Analysis of persistence also implies random locomotion. Compilation of the distribution of angles between steps reveals that the cells exhibit a predilection for turns around 30° and 150° on either side of the previous step. Time-lapse video recordings show that the cells are bi-polar with ruffling membranes at opposite poles. Ruffling, and hence locomotion, occurs alternately at one pole and then the other, which would account for the distribution of angles encountered. The mean speed of the cells was of the order of 3 μm min−1 including the time stopped and approximately twice this if the time stopped (state 0) is not included. The results obtained provide base-line data on the locomotory characteristics of MOS-11 cells locomoting in a 1·2 mg ml−1 collagen gel. It is now possible to study the role of various matrix components in cell locomotion. Such studies are of importance to embryology, wound healing, host defence mechanisms and the invasion of cancer cells.
Deficiency of density-dependent regulation of cell growth in the culture of skin fibroblasts from patients with mucolipidosis iii
Authors: Oohira Atsuhiko , Matsui Fumiko , Oki Takashi , Nogami Hiroshi
Published: April 2021

Cultured skin fibroblast cells were prepared from two patients with mucolipidosis HI (ML HI), which is a genetic disorder characterized by low activities of multiple lysosomal enzymes in fibro-blasts. Genetic complementation analysis of fused fibroblast hybrids revealed that the patients were classified in different complementation groups. Growth curves of fibroblasts of ML HI patients in culture were compared with those of fibroblasts of Sanfilippo’s syndrome patients as well as of the normal fibroblasts. Normal and Sanfilippo fibroblasts gave essentially the same sigmoid curve of cell growth. However, although both ML m cell lines grew at the normal rate in the initial logarithmic phase, they continued to proliferate actively even after the cultures reached confluency. This is the first report to demonstrate the deficiency of density-dependent regulation of cell growth in the culture of non-transformed cell types. Therefore, the culture of skin fibroblasts of ML HI patients may serve as a useful experimental model for investigating the regulation of cell proliferation in vitro.
Identification of calmodulin in Acetabularia: its distribution and physiological significance
Authors: Cotton Geneviève , Driessche Thérèse Vanden
Published: April 2021

In order to test whether calmodulin is present in Acetabularia, material was isolated from this alga, using the chlorpromazine affinity method. This resulted in the purification of a protein with the correct mobility on SDS–polyacrylamide gels. This protein reacted with anti-calmodulin in dot blots, using an immunogold, silver-enhanced method, so we conclude that calmodulin is present in the alga, although at a very low concentration (200–450 pg per gram wet weight).

The localization of calmodulin was studied, using fluphenazine fluorescence. The pattern is described; the chief feature being the concentration of fluorescence towards the tip during both the slow and the rapid growth phases. Fluorescence is also observed at the insertion points of hairs on the stalk and decreases in intensity between hairs of successive order. The apical fluorescence vanishes when cap formation begins. The localization of calmodulin parallels that of calcium studied by means of chlorotetra-cycline and aequorin.

Expecting inhibitors of calmodulin to produce physiological effects, we studied the growth of whole and anucleate Acetabularia in the presence of trifluoperazine (TFP). TFP severely inhibited growth and cap formation, as did lanthanum. A circadian rhythm of sensitivity to these inhibitors was found. The hypothesis is put forward that calcium and calmodulin are important during a critical photosensitive phase of the circadian cycle.
An actin-related protein inside pea chloroplasts
Authors: Mccurdy David W. , Williamson Richard E.
Published: April 2021

A pea chloroplast protein resembles vertebrate and algal actins by several chemical and immunological criteria. On two-dimensional polyacrylamide gels it migrated with a slightly lower relative molecular mass (Mr = 41 000) and slightly more basic isoelectric point than rabbit skeletal muscle actin. A monoclonal antibody to chicken gizzard actin reacted on immunoblots with rabbit skeletal actin, with Chara actin and with a 41000Mr band from pea chloroplasts. Pea and Chara bands of approximately 58 000Mr were also stained. A DNase I-affinity column that bound muscle actin also bound 41 000 and 58 000Mr chloroplast polypeptides. Similarities existed between enzymically and chemically generated fragments of the 41000Mr chloroplast polypeptide and rabbit muscle actin. The 41000 Mr protein was protected from degradation by thermolysin only in preparations of intact, but not ruptured, isolated chloroplasts, indicating that this protein resides within the outer envelope membrane of these organelles. It is concluded that a 41 000 Mr protein with major similarities to actin occurs inside pea chloroplasts, and that a 58000Mr protein with some similarities to actin also probably exists within chloroplasts.
Surface membrane clearing of receptor–ligand complexes in human blood platelets
Authors: Behnke O.
Published: April 2021

Human blood platelets were challenged sequentially in vitro with polyclonal anti-platelet antibodies and cationized ferritin. Both ligands bound to the surface membrane and were sequestered by internalization into a surface-connected membrane system (SCS) with a cleared surface membrane as the eventual result. Patching and capping of bound antibody preceded internalization, and platelets cooperated in the clearing process by adhering to each other at capped areas and by mutual covering up, a process dubbed platelet hugging. The internalization process was repeated upon challenge with the second ligand, the two ligands being sequestered as separate deposits in the SCS, mirroring the two cycles of internalization. Platelets were activated during the internalization process and tended to aggregate. In aggregates, surfaces exposed to the medium were cleared of ligand, which accumulated in the intercellular spaces and within the SCS of the aggregated platelets. Aggregation but not internalization and hugging was prevented by adenosine and adenosine monophosphate.
The role of reactive oxygen metabolites in lymphocyte-mediated cytolysis
Authors: Bishop C. J. , Rzepczyk C. M. , Stenzel D. , Anderson K.
Published: April 2021

To examine the possible role of reactive oxygen metabolites in lymphocyte-mediated cytolysis, the morphology of cell death following the exposure of cells to reactive oxygen metabolites in vitro was compared with the morphology of cell-mediated killing in vitro of tumour cells by natural killer (NK) cells. Ultrastructural examination of human tumour cells that were dying following incubation for 60 min with the oxygen metabolite generating systems, xanthine-xanthine oxidase or t-butylhydroperoxide, showed that cell death in both instances was exclusively by necrosis. It was unclear which oxygen metabolites were involved in killing. Cell death was not decreased by the addition of superoxide dismutase, a scavenger of the superoxide anion, to the xanthinexanthine oxidase mixture. Although the cells were not killed by incubation with 1 mM-hydro-gen peroxide, the addition of catalase, a scavenger of hydrogen peroxide, to the xanthinexanthine oxidase mixture significantly reduced cell death. The addition of scavengers for the hydroxyl radical to either the xanthine-xanthine oxidase mixture or t-butylhydroperoxide gave inconsistent protection.

In contrast, tumour cell killing mediated by natural killer cells was by apoptosis, a morphologically distinct mode of cell death with a different basic mechanism, indicating that reactive oxygen metabolites are not directly involved in lymphocyte-mediated cytolysis.
Isolation of an endocytic compartment from A431 cells using a density modification procedure employing a receptor-specific monoclonal antibody complexed with colloidal gold
Authors: Beardmore Julia , Howell Kathryn E. , Miller Karen , Hopkins Colin R.
Published: April 2021

Our objective was to isolate a prelysosomal compartment involved in receptor-mediated endo-cytosis in human epidermoid carcinoma (A431) cells. The isolation protocol involves density modification of endosome elements in A431 cells, caused by the receptor-dependent binding and internalization at 20 °C of colloidal gold-transferrin receptor antibody (B3/25) particles. The use of 125I-labelled gold–B3/25 provides a radioactive marker for the endosome compartment, the major peak being recovered at the bottom of a continuous sucrose gradient at a density of 1·23 g ml−1. Enzyme markers characteristic of other cytoplasmic compartments are present only in negligible amounts in this fraction and L-[35S]methionine-labelling of the cells indicates approximately a 200-fold enrichment of bilabelled gold–B3/25 versus protein. Electron microscopy of the endosome-rich fraction reveals that we have isolated a highly purified population of small gold-containing vesicles and tubules from which the transferrin receptor can be immunoprecipitated using the B3/25 antibody. Gel electrophoresis and fluorography of L-[35S]-methionine-labelled cells suggests that these elements contain a characteristic profile of approximately 10 major proteins of which three appear to be specifically enriched. In cells incubated with [125I]transferrin, 12% of the ligand sediments with the gold-labelled elements. We conclude, therefore, that the components we have isolated play a role in the intracellular processing of the transferrin-transferrin receptor complexes.
Quiescence and increased adhesion after mammalian DNA transfection of human fibrosarcoma cells
Authors: Nagy Adam , Baker Robert F.
Published: April 2021

When a population of human fibrosarcoma HT1080 cells was transfected with a mammalian expression vector and DNA pieces representing either the human whole genome or mouse bulk cDNA, there was a transient increase in the number of adhesive cells in the population. The number of cells with increased adhesion was proportional to the amount of transfected DNA; the increase occurred at a maximal frequency of between 10−4 and 10−5 per cell. Increased adhesion in these cells persisted for 9–12 days, corresponding to the period of highly efficienttransient transfection, and was accompanied by arrest in cell division. Transfection of non-mam-malian DNAs, reduction of transfected mammalian sequence length by restriction enzyme digestion, or omission of expression vector DNA did not permit these shifts in phenotype. The effects seen suggest that expression of specific transfected mammalian DNA sequences suppresses certain phenotypic characteristics in these transformed mammalian cells.
A monoclonal antibody recognizes a human nuclear protein resembling Xenopus oocyte nucleoplasmin
Authors: Lord Janet , Brown Geoffrey , Bunce Christopher , Nathan Paul
Published: April 2021

The monoclonal antibody 10BG2 (IgG3) was derived from a mouse immunized with human pre B cells. In immunofluorescence studies the antibody revealed a human nuclear-associated determinant, which in interphase cells was entirely restricted to the nucleus. In metaphase cells 10BG2 antigen was detected throughout the cytoplasm with intensified staining at the periphery of chromosomes. 10BG2 antibody stained all human normal and transformed cells tested. In contrast, the antibody did not stain mouse 3T3 cells or Chinese hamster ovary (CHO) cells. Electrophoresis under denaturing and non-denaturing conditions revealed the 10BG2 antigen to be a 130 ×103 to 140×103Mr protein with a subunit molecular weight of 29·5×103. This suggests the protein is at least a tetramer. On two-dimensional gels the 10BG2 antigen had a streaked appearance and separated into two isoelectric variants (pl 5·2, 5·4). The protein was also shown to be phosphorylated and thermostable, and remained in solution at pH 3·6. 10BG2 antigen was highly soluble in aqueous buffers and co-migrated on non-denaturing gels with the nucleosome-assembly protein, nucleoplasmin, purified from Xenopus oocytes. The similarity of 10BG2 antigen to nucleoplasmin is discussed.
Hydration, volume changes and nuclear magnetic resonance proton relaxation times of HeLa S-3 cells in M-phase and the subsequent cell cycle
Authors: Wheatley Denys N. , Inglis Marget S. , Foster Margaret A. , Rimington J. Ewen
Published: April 2021

Most mitotic HeLa cells divided into two daughter cells with half the volume of the parent; no additional reduction in volume was detectable during late telophase or the early part of G1. Synchronous growth throughout the next generation cycle was exponential, without an additional and sudden rise in volume being detectable as cells entered M-phase. Since the wet weight / dry weight ratio and the protein per unit mass of cells remained constant throughout the cycle, measurements based on volume changes accurately reflected growth parameters and cell water content.

Nuclear magnetic resonance (n.m.r.) t1 (spinlattice) proton relaxation times for intracellular water for M-phase cells measured at both 32 MHz (25°C) and 80MHz (37°C) in a Bruker spectrometer had the same values as normal interphase cells. Furthermore, cells arrested in metaphase by nitrous oxide or alkaloids gave the same t1 values. Artificial manipulation of cell hydration by adjustment of the tonicity of the external medium led to t1 values that correlated well with the level of intracellular water. On this basis, a 40% increase in hydration raised t1values by a factor of 1 ·29 at 32 MHz and 1 ·5 at 80MHz. This is considerably less than the increase by a factor of 1 ·9 in t1 time reported with the 40% rise in cell water accompanying mitosis, measured at 30 MHz and 25 °C under isotonic conditions by others. The protein-synthesizing ability of hydrated cells was reduced to only half the normal level after a doubling of intracellular water.

The data from these several different analyses, taken together, strongly indicate that the water content of mitotic cells is very similar to that of interphase cells, and that certain features unique to the mitotic phase of the cell cycle cannot be ascribed to an elevated (free) water content.
The microtubule-organizing complex and the Golgi apparatus are co-localized around the entire nuclear envelope of interphase cardiac myocytes
Authors: Kronebusch Paul J. , Singer S. J.
Published: April 2021

In most animal cells, the microtubule-organizing centre (MTOC) and the Golgi apparatus (GA) are co-localized on one side of the nucleus, an arrangement that allows these cells to acquire a functional polarity. An exception has been reported in the skeletal muscle myotube, where the MTOC and GA exhibit a circumnuclear distribution. We wished to determine if this unusual distribution of the MTOC and GA was peculiar to syncytial myotubes or reflected a pattern found in muscle cells generally. Immunofluorescence microscopic studies of cultured chicken skeletal muscle, cardiac muscle and gizzard smooth muscle cells were carried out using preimmune sera that recognized the pericentriolar material, anti-tubulin antibodies to label the MTOC, and fluorescent wheat-germ agglutinin to label the GA. These studies have shown that cardiac myocytes possess a circumnuclear distribution of their MTOC and GA as do skeletal myotubes, but smooth muscle cells exhibit the centrosomal MTOC and GA distribution found in most other cells. The circumnuclear MTOC/GA distribution therefore is associated with striated muscle cells. We also found that as embryonic cardiac myocytes pass through the cell cycle the microtubuleorganizing activity in these cells switches from a circumnuclear distribution in interphase to the conventional centrosomal location during mitosis. Thus, cardiac myocytes provide a rare example of mononucleated animal cells that do not display a centrosomal MTOC or a polarized GA, and also reveal a system in which the MTOC structure can be reversibly altered in a cell cycledependent manner.
Isolation and characterization of a mutant of Tetrahymena thermophila blocked in secretion of lysosomal enzymes
Authors: Hünseler Peter , Scheidgen-Kleyboldt Gerda , Tiedtke Arno
Published: April 2021

The development of a sensitive screening procedure for mutants of Tetrahymena thermophila blocked in secretion of lysosomal enzymes is described.

By means of this procedure a mutant blocked in secretion of lysosomal enzymes has been isolated. This sec− mutant, MS-1, is constitutively blocked in release of at least six lysosomal enzymes, under both nutrient and non-nutrient conditions. MS-1 possesses, bound within the cell, the same amount of active lysosomal enzymes as the wild type. During starvation in media of low ionic strengh MS-1 develops a highly vacuolated phenotype. This phenotype is caused by the sec− allele. It is reversed to a normal cell shape when the mutant is transferred to isotonic medium.

The sec− mutant MS-1 contains mucocysts and is capable of inducing exocytosis of these secretory organelles, suggesting that Tetrahymena possesses at least two independent protein-secreting organelles.

Key words: Tetrahymena thermophila, lysosomal enzymes, protein secretion, secretory mutants.
Analysis of the temperature-sensitive period of the short-1 mutation affecting stomatogenesis in Paramecium tetraurelia
Authors: Tam Lai-Wa , Ng Stephen F.
Published: April 2021

Reduction in the length of the oral apparatus produced by the temperature-sensitive mutation short-1 (shl) involved suppressed growth of the oral primordium in all stages of development. Temperature shift-up and heat-shock experiments revealed that the temperature-sensitive period of this mutation coincided with nearly the entire stomatogenic phase (stages 1–6) in sexual reproduction. Low- and high-sensitivity phases were noted, corresponding to the periods of slow (stages 1 and 2) and rapid (stage 3 to stage 6) elongation of the oral primordium, respectively. The action of shl is thus concentrated after stage 2. The mutation hypothetically results in defective membrane growth and extension in the oral primordium, leading to restriction in incorporation of basal bodies into the developing membranelies.
A glycoprotein rapidly labelled with [14C]glucose present in a murine melanoma cell line
Authors: Bramwell M. E.
Published: April 2021

If a murine melanoma cell line PG19 is metabolically labelled with [14C]glucose for a short time, one particular membrane component incorporates more radioactivity than the rest. This is a glycoprotein of about Mr 100 000, which is not preferentially labelled in non-malignant variants of the melanoma or in suppressed hybrid cells formed between PG 19 and mouse fibroblasts.
Organization of the boar spermatozoan plasma membrane: Evidence for separate domains (subdomains) of integral membrane proteins in the plasma membrane overlying the principal segment of the acrosome
Authors: Peterson R. N. , Gillott M. , Hunt W. , Russell L. D.
Published: April 2021

Indirect immunofluorescence microscopy and freeze-fracture have been used to identify over-lapping subdomains at the peripheral rim of the sperm-head plasma membrane (PM) and the margin of the outer acrosomal membrane (OAM) comprising the principal segment of the acrosome of the boar spermatozoon. An array of ridge-like structures (spaced 12–16 nm centre-to-centre), originally observed on the OAM by Aguas & Pinto da Silva, lies just beneath an area of the PM that is sparsely populated with large intra-membranous particles compared to that of other regions of the head PM. This region has a high specificity for the lectin aracbis hypogaea (peanut agglutinin). We suggest that the OAM at the rim of the sperm head may be rich in acidic phospholipids and that the close apposition of this membrane with a region of the PM relatively poor in integral membrane proteins may provide sites for initiating the acrosome reaction.