bluuebunny/crossref_metadata_2025 huggingface dataset:
Recent findings on oogenesis of Drosophila melanogaster: I. Ultrastructural observations on the developing ooplasm
Authors: Giorgi F. , Jacob J.
Published: April 2021

The ultrastructure of the developing ooplasm has been examined in 2–3-day-old flies of Drosophila melanogaster with a view to clarifying the origin of the yolk platelets.

Previtellogenic ovarian chambers have been shown to exhibit a number of cytolysosomes; these are first formed in the nurse cells and are later brought to the ooplasm after having passed through the ring canals.

Just prior to the beginning of vitellogenesis some of the cytolysosomes in the ooplasm acquire in places a yolk-like appearance. As vitellogenesis commences, in ‘early stage-8 chambers, the central ooplasm comes to exhibit a number of inclusions which combine in themselves features of both yolk platelets and cytolysosomes.

The onset of vitellogenesis at stage 8 is marked by the appearance of several structural entities on the plasma membrane of the oocyte. At this site, there appear a number of depressions or pits in between the microvilli. Deeper in the cortical ooplasm there are also present a number of coated vesicles and tubules. While the former exhibit a typical threelayered structure similar to that of the pits, the tubules lack the outer spiked layer.

Mature yolk platelets appear to be made up of three major components. These are a central homogeneous dense body, an outer superficial layer with a number of small spherules and a rounded body therein, and an external limiting membrane.

Instances of fusion between vesicles of sizes progressively larger have been observed with frequency. This observation is discussed in relation to the formation of yolk platelets by means of pinocytotic uptake. A contribution made by the nurse cells to the formation of the yolk platelets is also taken into account.
Preleptotene chromosome condensation stage in human foetal and neonatal testes
Authors: Lucian J. M. , Devictor Monique , Stahl A.
Published: April 2021

A preleptotene stage of chromosome condensation analogous to that already described in various plants and in the oocytes of several animal species has been observed in the human foetal testis. Contrary to what has been previously described, this stage in the testis is not followed by decondensation leading to leptotene filaments. This observation underlines the problem of the precise significance of this stage and its relation to initiation of meiosis. It is suggested that meiosis may be initiated during this condensation phase and that the male germ cell, despite its XY chromosome constitution, tends to evolve towards meiosis. This proposal pleads in favour of both the role of somatic cells in the inhibition of meiosis in the male foetus and the role of environmental factors rather than genetic constitution of the germ cell in meiotic induction.
Immunohistochemical studies of vitellogenin during embryogenesis in the cockroach Blattella germanica
Authors: Tanaka Akira
Published: April 2021

The fate of vitellogenin, vitellogenic female-specific protein, during embryogenesis in the German cockroach was investigated by an immunofluorescent technique. The period of the embryonic development of this insect is about 25 days at 25 ± 1 °C, and it has been divided into 18 stages (Tanaka, 1976).

Vitellogenin-specific fluorescence was evenly distributed among yolk spheres until stage 4. The intensity of the fluorescence of each yolk sphere, however, began to be uneven at stage 5, and the unevenness became more distinct in stages 6 – 7 to reach a mosaic appearance with respect to fluorescence. After stage 8, the mosaic appearance disappeared, and the intensity of fluorescence of the yolk region decreased on the whole as the stage advanced.

In vitellophages, no specific fluorescence was observed until stage 6. Very strong fluorescence, however, became observable in some vitellophages at stage 7. All vitellophages became fluorescent after stage 8. Some vitellophages, however, lost the fluorescence after stage 12, though the strong fluorescence still persisted in others until a fairly late stage when the cells became shrunken.

The specific fluorescence was observed in the yolk region only until stage 10. The nuclei of the body region of the embryo, however, began to fluoresce very strongly at stage 11 when dorsal closure had occurred. It persisted during stages 11 – 13, and abruptly declined after stage 14. The cytoplasm of the body region also had comparatively weak fluorescence at these stages. In the appendageal region including the antennae and pleuropodia, the specific fluorescence was not observed at all either in the nuclei or cytoplasm during development.
Differentiation of primordial germ cells in the embryonic development ofThermobia domestica. Pack.(Thysanura):an ultrastructural study
Authors: Klag Jerzy
Published: April 2021

The primordial germ cells (PGCs) of Thermobia domestica undergo some morphological changes during the embryonic development. Most conspicuous are the changes in the ultrastructure of the nucleus, whose envelope shows a high degree of activity. Two types of vesicles bleb off from the nucleus; the ones with the light interior are called the accessory nuclei, the others, with electron-opaque contents, have been termed the dense bodies. The nucleolus, initially clustered at the nucleus centre, undergoes dispersion and assembles again towards the end of embryonic development. At the same time, the sex differentiation of PGCs takes place. It is preceded by an increase in the activity of Golgi complexes and in the volume of lysosomes and lamellar bodies, the latter giving rise to lipid droplets. At the early stages of postembryonic development, preoogonia and prespermatogonia can readily be distinguished. Preoogonia have a wavy-surfaced nucleus and their cytoplasm contains dense bodies. In prespermatogonia, the nucleus is spherical with smooth envelope and there are no dense bodies in the cytoplasm. Throughout the period studied there occur nucleolus-like bodies and nuage material considered to be the germ-cell determinants in this species.
Ontogeny of lens crystallins in marine cephalopods
Authors: Brahma Samir K.
Published: April 2021

The ontogeny of the lens crystallin antigens throughout development of three marine cephalopod embryos, Loligo vulgaris, Sepia officinalis and Octopus vulgaris has been investigated using the indirect immuno-cytochemical staining against respective homologous anti-total lens protein antiserum.

The cellular mechanism of lens development appears to be the same in all species investigated and so does the ontogeny of the lens crystallins. The first positive immunofluorescence reaction appears simultaneously and in equal intensities over the lens and lentigenic area confirming the relationship between the two.
Electron microscopic studies on the structure of motile primordial germ cells of Xenopus laevis in vitro
Authors: Heasman Janet , Wylie C. C.
Published: April 2021

Primordial germ cells (PGCs) of Xenopus laevis have been isolated from early embryos and kept alive in vitro, in order to study the structural basis of their motility, using the transmission and scanning electron microscope. The culture conditions used mimicked as closely as possible the in vivo environment of migrating PGCs, in that isolated PGCs were seeded onto monolayers of amphibian mesentery cells.

In these conditions we have demonstrated that: (a) No significant differences were found between the morphology of PGCs in vitro and in vivo.(b) Structural features involved in PGC movement in vitro include (i) the presence of a filamentous substructure, (ii) filopodial and blunt cell processes, (iii) cell surface specializations. These features are also characteristic of migratory PGCs studied in vivo.(c) PGCs in vitro have powers of invasion similar to those of migrating PGCs in vivo. They occasionally become completely surrounded by cells of the monolayer and, in this situation, bear striking resemblance to PGCs moving between mesentery cells to the site of the developing gonad in stage-44 tadpoles.

We conclude that as far as it is possible to assess, the behaviour of isolated PGCs in these in vitro conditions mimics their activities in vivo. This allows us to study the ultrastructural basis of their migration.
In vivo and in vitro studies on the hypoblast and definitive endoblast of avian embryos
Authors: Sanders E. J. , Bellairs Ruth , Portch P. A.
Published: April 2021

An unusual example of the invasion of one tissue by another occurs during gastrulation in the chick embryo when the definitive endoblast becomes inserted into the hypoblast. The two tissues were examined morphologically by SEM and TEM. They resemble each other in being of an epithelial type, though neither possesses a basal lamina. The definitive endoblast cells are flatter than the hypoblast cells and more closely attached to one another. When they were explanted in hanging drop cultures, the two tissues were found to exhibit differences in their behaviour. In comparison with the definitive endoblast, the hypoblast cells attached more readily to the glass, produced larger ruffle membranes, moved more rapidly, showed poorer contact-inhibition of locomotion and showed a greater tendency to break away from the main explant.

When a hypoblast explant was confronted with a definitive endoblast explant, the hypoblast cells became displaced by the definitive endoblast. The hypoblast explant tended to fragment into smaller groups of cells, many of which migrated around the definitive endoblast, thus mimicking the situation in vivo. Control experiments comprised confronting hypoblast with hypoblast, hypoblast with somites, definitive endoblast with definitive endoblast, and definitive endoblast with somites. The hypoblast explants behaved in a consistent manner, always fragmenting when coming into contact with cells from a confronting explant. The definitive endoblast explants showed more contact inhibition of locomotion when confronted with definitive endoblast or with somites than when confronted with hypoblast. It is suggested therefore that the ability of the hypoblast cells to separate from one another may play an important role in the penetration of the hypoblast by the definitive endoblast both in vitro and in vivo.
Différenciation cytophysiologique régionale de l’épithélium uropygien chez l’embryon de canard Anas platyrhynchos
Authors: Bride Jacqueline
Published: April 2021

The uropygial gland (preen gland), an epidermal derivative, is a bilobed, lipid-secreting gland located over the base of the tail of most birds.

In the duck embryo Anas platyrhynchos, the internal branching morphogenesis of preen gland is set up at the 17th day of incubation. Each glandular lobe is made of numerous epidermal columns each of which is terminated by a bulb or end-bud. The functional differentiation of the end-buds and the development of the cellular columns into collecting ducts were investigated. The ductal epithelium, separated from the mesoderm by a continuous basal lamina, is keratinized just as in normal embryonic avian epidermis. The features which indicate glandular differentiation in the end-buds were described. Lipogenesis results from progressive cellular differentiation characterized by proliferation and development of smooth membranes. The direct ecto-mesodermal contacts, which were observed at the endbuds after the establishment of morphogenetic pattern and before the onset of glandular secretory activity, suggest that a new interaction mechanism would be required to initiate the functional differentiation.
Regional differences in mesenchymal cell morphology and glycosaminoglycans in early neural-fold stage rat embryos
Authors: Morriss Gillian M. , Solursh Michael
Published: April 2021

Rat embryos with two to four pairs of somites (day 9 of gestation) were examined by scanning electron microscopy and Alcian blue staining. The neural folds, which represent only future brain region at this stage, form a pair of elongated hemispheres with a deep neural groove between them. In transverse section the neural ectoderm is biconvex; the cranial mesenchyme cells beneath them are widely separated by extracellular matrix (ECM) and are joined to each other and to the ectodermal basement membrane by fine cytoplasmic processes and strands of ECM material. In contrast, mesenchyme cells close to the primitive streak are closely packed, having broad areas of surface contact and only small amounts of ECM.

The nature and distribution of ECM, cell surface, and basement membrane glycosaminoglycans (GAG) were investigated by staining with Alcian blue at specific pH values in combination with enzyme pretreatments, and at various critical electrolyte concentrations. The results indicate that the GAG of the ectodermal basement membrane, mesenchymal ECM and mesenchymal cell surfaces are in continuity with each other and consist largely of hyaluronate and chondroitin sulphates.

Differences in morphology and histochemistry of neural fold and primitive streak regions are discussed in relation to their possible morphogenetic significance.
The role of the cell surface in the migration of primordial germ cells in early chick embryos: effects of concanavalin A
Authors: Lee H. , Karasanyi N. , Nagele R. G.
Published: April 2021

Effects of concanavalin A (Con A) on the morphology and migration of primordial germ cells (PGCs) in stage-6 to -12 chick embryos were investigated. Con A, at a sublethal dose (10 μg/ml), inhibited migration of PGCs from the germinal crescent area to other parts of the embryo. Affected PGCs were more rounded without the usual cytoplasmic extensions, but the integrity of other structures was unaffected. Nearly identical results were obtained with another lectin, wheat germ agglutinin (10 μg/ml). Histochemistry using Con A-horseradish peroxidase revealed that PGCs in control embryos had a thin, rather uniform layer of extracellular coat material (ECM). Con A appeared to alter the distribution of ECM on PGCs, i.e. some parts of the cell surface were devoid of any detectable ECM, while others had small, scattered patches of ECM. Con A effects were alleviated by a-methyl-D-mannoside. Overall results of the present study indicated that the observed inhibition of PGC migration in early chick embryos is a consequence of Con A-induced alterations of cell surface properties.
Cell proliferation and cell density of mesenchyme in the maxillary process and adjacent regions during facial development in the chick embryo
Authors: Minkoff Robert , Kuntz Amy J.
Published: April 2021

Cell proliferation, as measured by DNA labeling indices was analyzed during the early development of the maxillary process. Chick embryos were labeled with [3H]thymidine for .1 h and processed for autoradiography. The percentage of labeled mesenchymal cells was determined within delineated areas in the maxillary processes and in adjacent regions. Analysis of labeling indices in each of the areas at successive stages of development demonstrated a pattern of declining rates of cell proliferation with advancing developmental age. Cell proliferation in adjacent regions declined earlier and, in some instances, faster than it did in the maxillary process.

Cell density was measured in the maxillary process and the roof of the stomodeum and was found to be higher in the maxillary process throughout the period studied. Cell density and cell proliferation data were analyzed with reference to the operation of ‘density-dependent inhibition’ of growth as a regulatory mechanism for the observed changes. ‘Density-dependent inhibition’ of growth was not a satisfactory explanation for the observed differences between the maxillary process and adjacent regions.
Ultrastructural study of the development of the auditory tympana in the cricket Teleogryllus commodus (Walker)
Authors: Ball E. E. , Cowan A. N.
Published: April 2021

The cuticle in the tympanal area of immature crickets, Teleogryllus commodus (Walker), is ultrastructurally indistinguishable from that elsewhere on the prothoracic leg. It is only in the pharate adult that changes associated with development of the tympana first appear. In pharate adults and adults the external layer of the tympana consists of a layer of electron-dense material overlying a layer where the electron-dense material is interspersed with cuticle in which the bundles of microfibrils are coarser and more loosely arranged than elsewhere in the leg. The innermost portion of the tympana consists of this same type of cuticle without the electron-dense material. Associated with the appearance of the electron-dense material in the tympana of the pharate adult is a change in the toluidine blue staining properties from blue to deep purple. The reaction of the tympana in acid and base is consistent with their being composed of chitin. There are no major deposits of resilin in the tympana. In the first few days following the imaginal ecdysis the posterior tympanum and underlying trachea come into tight apposition due to the withdrawal of the epidermal cells. The epidermal cells do not withdraw from beneath the anterior tympanum. The surrounding non-tympanal cuticle continues to thicken for several weeks with the result that in the mature adult the posterior tympanum serves as an acoustic window in the thick cuticle of the leg. The functional significance of the anterior tympanum has not been established.
The kinetic behaviour of the cranial neural epithelium during neurulation in the rat
Authors: Tuckett Fiona , Morriss-Kay Gillian M.
Published: April 2021

The kinetic behaviour of the cranial neuroepithelial cells of rat embryos during neurulation is described. Serial transverse sections of 4-, 8-, 12- and 16-somite-stage embryos show that differential mitosis does not play a part in the mechanisms responsible for effecting cranial neural tube closure. A constant cell number is found in the midbrain/hindbrain neural epithelium during all four stages; the mitotic spindle axes are orientated parallel to the long axis of the embryo, so that increase in cell number occurs in this direction only.

Growth is only expressed by an expansion in the volume of the forebrain, which projects rostral to the notochordal tip. [3H]thymidine studies (using an in vitro culture technique) show no significant variation in the cell cycle time between the forebrain and the midbrain/ anterior hindbrain neural epithelium. It is suggested that the neural epithelium is a fluid structure whose overall shape is strictly controlled while the cells within it flow towards and into the rapidly expanding forebrain.
Development in vitro of the female germ cells of the polychaete Ophryotrocha labronica
Authors: Emanuelsson Hadar , Anehus Siw
Published: April 2021

In the polychaete Ophryotrocha labronica each oocyte is during its growth period associated with a single nurse cell. The fact that the oocyte-nurse cell pairs occur isolated in the female coelom makes them easily removable for analysis of their developmental ability in vitro. Using Dulbecco’s modified Eagle medium supplemented with amino acids, nucleosides, foetal calf serum and sea water, we have managed to support development in vitro of germ cell pairs from early and mid-oogenesis until maturation of the oocyte, when the nurse cell degenerates and the oocyte enters meiotic metaphase. Radiolabelling of germ cells in mid-oogenesis with tritiated amino acids and uridine during the first day of incubation indicates normal development with synthesis of RNA and protein, and pulses two days later verify a continued normal protein synthesis and yolk formation. The investigation confirms autosynthesis of yolk proteins in the germ cells of this species and indicates a leading role of the nurse cell in the process.
An Investigation of Renal Function during Histogenesis in the Late Foetal and Neonatal Rat by means of the Uptake and Autoradiographic Localization of Radioactive Potassium
Authors: Bearn J. G. , Harris F. T. C.
Published: April 2021

A technique has been developed for studying the uptake of K42 by the foetal rat kidney. Using this technique the exchange rate for K42 of the foetal and neonatal metanephros relative to the foetal and neonatal liver has been investigated. It was found that the metanephros of the 20- and 21-day foetus shows a rate of exchange which is twice that of the liver, whereas the neonatal metanephros shows a rate of exchange which is less than half that of the liver. The results of this investigation demonstrate that the ratio of K42 uptake by the metanephros of the 20- and 21-day rat foetus, relative to that of the foetal liver, is one-quarter of that reported for the adult rat. An autoradiographic study of the distribution of K42 in the foetal kidney has shown that histological localization of the isotope in the proximal and distal tubules, found in the adult, does not occur.
Half-lives of enzyme activities in an l5178y mouse leukaemic cell
Authors: Bosmann H. B.
Published: April 2021

L5178Y cells (mouse leukaemic cell lines) were grown in Fischer’s medium and were utilized in the logarithmic phase of growth. Cells were incubated at 37 °C in 100 pg per ml of cycloheximide. This level of cycloheximide inhibited all cytoribosomal protein synthesis after 30 min incubation. After 7·0 h incubation with 100 μg per ml of cycloheximide the cell number was, in arbitrary units, 1·07 ±0·001 (control: 1·63 ±0·03), the protein content i6i±4pg per cell (control: 181 ± 1) and the percentage viable cells 17 ±2 (control: 98 ±1). Cells were removed at intervals from the incubation in 100 μg per ml of cycloheximide, extracted with 0·1% Triton X-100, and assayed for various enzyme activities. From these data half-lives, stability times, and decay rates were calculated for each enzyme activity.

Lysosomal enzyme activities, in particular the jV-acetyl hexosaminidases, were found to have long half-lives and stability and slow decay rates. Proteolytic enzyme activities were characterized by relatively long half-lives and stability times. The membrane marker enzyme activities, especially UDPase, were characterized by extremely short half-lives, rapid decay rates, and no stability time. Mitochondrial enzyme activities had relatively short stability time and half-lives and rather rapid decay rates. The decay occurring at late time intervals was thought to be a consequence of the death phase of the incubated cells as opposed to normal cellular decay.
Effect of method of cell isolation on the metabolic activity of isolated rat liver cells
Authors: Lipson L. G. , Capuzzi D. M. , Margolis S.
Published: April 2021

Rat liver cells isolated with a tissue press, by tetraphenylboron (TPB) chelation, or by hyaluronidase and collagenase digestion were compared as to morphology, cell yield, and biosynthetic activity. The cells were intact by light microscopy; ultrastructural changes were present on electron-microscopic examination of all cell types except those prepared by a modified enzyme incubation method. TPB chelation gave the largest and enzyme techniques the smallest yield of cells. All cell types incorporated labelled amino acids into cellular protein; however, amino acid incorporation was greatest in cells isolated by the revised enzyme technique. Only enzyme and mechanical cells incorporated acetate into cellular lipid. Cofactor supplementation was not required in the modified enzyme cells. Acetate incorporation was more sensitive to preincubation than was amino acid incorporation. Calcium, which was required to prevent aggregation of enzyme cells, inhibited amino acid incorporation moderately and acetate incorporation completely in mechanical cells.
Contact-dependent transfer of a lysosomal enzyme from lymphocytes to fibroblasts
Authors: Bou-Gharios George , Moss Jill , Partridge Terry , Abraham David , Olsen Irwin
Published: April 2021

In this study we have examined the mechanism underlying the contact-mediated transfer of a lysosomal enzyme from lymphocytes to fibroblasts in culture. We found that although antibody against the mannose 6-phosphate lysosomal targetting receptor (MPR) completely inhibited fibroblast uptake of the lysosomal enzyme β-glucuronidase (Gus) from the culture medium, it had no effect on the transfer of the enzyme from normal lymphocytes. In contrast, the presence of antibody that prevented the adhesion of the lymphocytes to the fibroblasts inhibited Gus acquisition but had no effect on endocytosis. Immunogold electron microscopy of the contact site between the two types of cell showed that the transfer of Gus involved uncoated vesicles localized near the cell surface of the fibroblast at sites of contact with the lymphocytes. The acquired lymphocyte enzyme was shown to be transported to the fibroblast lysosomes.
Different patterns of α-tubulin post-translational modification in ovarian nutritive tubes of two hemipteran insects
Authors: Harrison Alistair , Stebbings Howard , Hyams Jeremy S.
Published: April 2021

Usage of the tyrosinated, detyrosinated and acetylated forms of α-tubulin in ovarian nutritive tube microtubules of the hemipterans Oncopeltus fasciatus and Notonecta glauca glauca was investigated by immunofluorescence microscopy of frozen sections of ovarioles with isotype-specific antibodies. In Oncopeltus, nutritive tubes at all stages of development contained tyrosinated α-tubulin and showed only a weak reaction to antibodies to the detyrosinated and acetylated forms. In Notonecta, tyrosinated β-tubulin was confined to a zone around the periphery of functional nutritive tubes; the body of these tubes, and the older, redundant, nutritive tubes stained strongly for both the detyrosinated and acetylated isotypes. The difference in isotype usage between the two species was confirmed by immunoblotting of 2-D gels of ovariole extracts. The results are consistent with the different time-course of oogenesis, and hence the longevity of the nutritive tube microtubules, in the two insects. A model for the insertion of new microtubules into nutritive tubes as they grow is proposed.
Organization of talin and vinculin in adhesion plaques of wet-cleaved chicken embryo fibroblasts
Authors: Feltkamp Constance A. , Pijnenburg Marian A. P. , Roos Ed
Published: April 2021

We have studied the fine structure of adhesion plaques in chicken embryo fibroblasts (CEF) and visualized the localization of vinculin and talin using immunoelectron microscopy on CEF opened by ‘wetcleaving’. This procedure, performed with nitrocellulose on cells grown on electron microscope grids, cleaved the CEF close to the inner face of the ventral membrane or at a slightly higher level through the cytoplasm. In the resulting preparations, adhesion plaques were identified by their localization at the end of microfilament bundles and by their density of vinculin and talin. The plaques showed a substructure of moderately electron-dense parallel bands that were interconnected. Both the parallel bands as well as the interconnecting threads showed a high density of vinculin and talin labels, whereas neither the surrounding membrane cytoskeleton nor the overlaying bundled microfilaments were labeled. In stereomicrographs, we observed no difference between the distances from vinculin or talin label, respectively, to the plasma membrane. In early spreading cells, vinculin and talin were found to be deposited simultaneously in fine radiating streaks that covered rather large parts of the ventral membrane at areas of close contact with the substratum. These streaks, which were initially over-layed by an isotropic cytoskeletal network without filament bundles, were the apparent precursors of later formed adhesion plaques. These observations suggest that there are no separate layers of talin and vinculin, but rather that adhesion plaques consist of a dense network of talin and vinculin. The observations strongly support the model proposed by Bendori et ?l. (1989), J. Cell Biol. 108, 2383-2393, that was based on the location of vinculin- and talin-binding sites in the vinculin molecule.
Ectopic expression of an A-type lamin does not interfere with differentiation of lamin A-negative embryonal carcinoma cells
Authors: Peter M. , Nigg E. A.
Published: April 2021

The nuclear lamina is an intermediate filament-type network underlying the inner nuclear membrane. It is believed to be important for nuclear envelope integrity and the organization of interphase chromatin. On the basis of biochemical properties and sequence criteria, vertebrate lamin proteins are classified as either A- or B-type. While B-type lamins are expressed in almost all cell types, no A-type lamins are present in early vertebrate embryos or undifferentiated embryonal carcinoma cell lines. Intriguingly, expression of A-type lamins occurs concomitant with cell differentiation and embryonic development. These findings have led to the hypothesis that A-type lamins might play a role in establishing or stabilizing cell-type specific differences in nuclear organization, which in turn might relate to the developmental potential of a cell. To test this hypothesis, we have stably expressed chicken lamin A in undifferentiated murine embryonal carcinoma (P19) cells, and examined the consequences of ectopic lamin A expression for the differentiation state and potential of these cells. Our results demonstrate that the P19 cells, although normally devoid of lamin A, properly incorporate and process chicken lamin A. Moreover, the stably transfected cell lines maintain the properties of undifferentiated cells, demonstrating that expression of lamin A does not directly induce differentiation. Conversely, when exposed to retinoic acid, an inducer of differentiation, lamin A-expressing P19 cells are able to differentiate normally. Taken together, our results suggest that unscheduled expression of A-type lamins is not sufficient to deregulate cell differentiation programs. The implications of these findings for the possible role for lamin A expression during development are discussed.
Arrangement of cellular fibronectin in noncollagenous fibrils in human fibroblast cultures
Authors: Dzamba Bette J. , Peters Donna M. Pesciotta
Published: April 2021

The assembly of fibronectin into fibrils was examined by high-voltage immunoelectron microscopy in subconfluent cultures of ascorbate-treated human skin fibroblasts. Cells grown in the presence of ascorbic acid for 24, 48 or 72 h were labeled with Ist-9, a monoclonal antibody specific for the EIIIA site in fibronectin, and polyclonal antibodies to type I collagen. Cells were then labeled with goat antimouse IgG and goat anti-rabbit IgG coupled to 5 or 18 nm colloidal gold beads. Our results show that by 24 h, fibronectin is observed in fibrils in the extracellular matrix. The majority of fibronectin in fibrils does not co-localize with type I collagen. Morphometric analysis of the distance between EIIIA sites in fibronectin fibrils (<l2nm in diameter) show that the EIIIA sites appear to be spaced approximately 84 nm apart. The distance of 84 nm suggests that fibronectin is fully extended in fibrils and that the amino termini of adjacent fibronectin dimers overlap by 20 nm. As fibronectin fibrils become thicker, the average distance between EIIIA sites in fibronectin dimers decreases to 42 nm. This decrease in the distance between EIIIA sites may be due to a staggering of fibronectin dimers within the fibril as the fibril matures.
Changes in relative concentrations of the hemoglobin components in Paramecium caused by cell growth and temperature of the culture
Authors: Usuki Itaru , Hino Akihiro
Published: April 2021

The Hb (hemoglobin) content of Paramecium has been known to maintain a constant level even when the cells are exposed to different conditions. However, the relative concentrations of the Hb components in the stocks of P. caudatum, P. multimicro-nucleatum and P. jenningsi were found to change during cell growth from logarithmic young phase to stationary growth phase even when the culture was maintained at a constant temperature of 18 or 27°C. The temperature of the culture also affected the relative concentrations of the Hb components in a cell. A large part of the growth-dependent and temperature-dependent change was attributable to a variation in the amounts of two or three major Hb components, and the variation in other minor components was almost negligible. At the time of cell growth and of a shift in temperature, the major Hb components in a cell respond in opposite ways from each other. These components differed in several properties: one of them showed a faster migration by PAGE, a smaller molecular mass and a lower pl value than another.
Altered response of progeria fibroblasts to epidermal growth factor
Authors: Colige A. , Nusgens B. , Lapiere CH. M.
Published: April 2021

The Hutchinson-Gilford syndrome (progeria) is a rare disorder in childhood characterized by premature and accelerated aging. This study reports the effect of a potent growth factor, EGF, on the proliferative capacities and extracellular matrix macromolecules and collagenase expression of two strains of progeria skin-derived cells. At low population doubling levels (PDL<IO), confluent cultures of progeria fibroblasts made quiescent by lowering the concentration of serum in the medium did not respond to EGF while the mitotic activity of normal PDL-matched fibroblasts was almost maximally restored upon addition of EGF. No obvious difference between normal and low PDL progeria fibroblasts was observed in the number and in the affinity of the receptors measured by [125I]EGF binding. The synthesis of collagen and non-collagen proteins was similar in normal and affected cells at low and high serum concentration and both types of cells re sponded to EGF by a specific inhibition of collagen synthesis. Besides a normal level of mRNA coding for type I and type III collagens, collagenase and laminin, progeria fibroblasts expressed a high level of elastin and type IV collagen mRNA. Like normal fibroblasts, progeria cells responded to EGF by a decrease in the level of mRNA for fibrillar collagens and elastin. In contrast, a complete lack of response to EGF was observed for collagenase mRNA whereas the expression of this enzyme was strikingly induced by EGF in normal PDL-matched cells. The abnormal expression of type IV collagen was not significantly modified by EGF. At PDL>IO, progeria cells exhibited features of senescence. A significant reduction of collagen synthesis was observed and no further inhibition by EGF was recorded.
Okadaic acid induces golgi apparatus fragmentation and arrest of intracellular transport
Authors: Lucocq J. , Warren G. , Pryde J.
Published: April 2021

The specific phosphatase inhibitor okadaic acid (OA) induced fragmentation of the Golgi apparatus in interphase HeLa cells. Immunoelectron microscopy for galactosyltranferase identified a major Golgi fragment composed of a cluster of vesicles and tubules that was morphologically indistinguishable from the ‘Golgi cluster’ previously described in mitotic cells. The presence of homogeneous immunofluorescence staining for galactosyltransferase in OA-treated cells also suggested that isolated Golgi vesicles, previously found in mitotic cells, existed along with the clusters. After removal of OA, both clusters and vesicles appeared to participate in a reassembly pathway that strongly resembled that occurring during telophase.

OA also induced inhibition of intracellular transport, another feature of mitotic cells. OA treatment prevented newly synthesised G protein of vesicular stomatitis virus (VSV) from acquiring resistance to endoglycosidase H and from arriving at the cell surface. In addition, fluid phase endocytosis of horseradish peroxidase (HRP) was reduced to less than 10% of control values. All these effects were dose-dependent and reversible.

OA should be a useful tool to study the Golgi division and membrane traffic.
Human immunodeficiency virus type 1 protease microinjected into cultured human skin fibroblasts cleaves vimentin and affects cytoskeletal and nuclear architecture
Authors: Hüner B. , Shoeman R. L. , Traub P.
Published: April 2021

In human skin fibroblasts microinjected with purified human immunodeficiency virus type 1 protease (HIV-1 PR), stress fibers were lost and alterations in nuclear morphology and condensation of nuclear chromatin were observed. Thereafter, the vimentin intermediate filament (IF) network collapsed. No effect was seen on the microtubules. While complicated by loss of affected cells from the substratum, a minimum estimate of the proportion of cells demonstrating these effects is 50%. Observation of single cells demonstrated that these effects were largely irreversible and were steps leading to the death of the HIV-1 PR-injected cells. After microinjection of various dilutions of the HIV-1 PR, it was observed that the changes in nuclear morphology and chromatin condensation were detectable under conditions where little or no effect was observed on both stress fibers and the IF network. Proteins of cells labelled with [35S]methionine and microinjected with either HIV-1 PR or BSA were subjected to two-dimensional gel electrophoresis. The major differences in the gel patterns were a diminution in the amount of vimentin and the appearance of novel products comigrating with cleavage products obtained after treatment of vimentin with HIV-1 PR in vitro. Thus, the HIV-1 PR is capable not only of cleaving IF subunit proteins in vivo, but also can catalyze alterations in other cellular structures.
Nature and origin of gap filaments in striated muscle
Authors: Trombitás K. , Baatsen P. H. W.W. , Kellermayer M. S. Z. , Pollack G. H.
Published: April 2021

Immunoelectron microscopy was used to study the nature and origin of ‘gap’ filaments in frog semiten-dinosus muscle. Gap filaments are fine longitudinal filaments observable only in sarcomeres stretched beyond thick/thin filament overlap: they occupy the gap between the tips of thick and thin filaments. To test whether the gap filaments are part of the titin-filament system, we employed monoclonal antibodies to titin (T-ll, Sigma) and observed the location of the epitope at a series of sarcomere lengths. At resting sarcomere length, the epitope was positioned in the I-band approximately 50 nm beyond the apparent ends of the thick filament. The location did not change perceptibly with increasing sarcomere length up to 3.6 μm. Above 3.6μm, the span between the epitope and the end of the A-band abruptly increased, and above 4 μm, the antibodies could be seen to decorate the gap filaments. Between 5 and 6μm, the epitope remained approximately in the middle of the gap. Even with this high degree of stretch, the label remained more or less aligned across the myofibril. The abrupt increase of span beyond 3.6 μm implies that the A-band domain of titin is pulled free of its anchor points along the thick filament, and moves toward the gap. Although this domain is functionally inextensible at physiological sarcomere length, the epitope movement in extremely stretched muscle shows that it is intrinsically elastic. Thus, the evidence confirms that gap filaments are clearly part of the titin-filament system. They are derived not only from the I-band domain of titin, but also from its A-band domain.
Activation of Dona sperm motility: phosphorylation of dynein polypeptides and effects of a tyrosine kinase inhibitor
Authors: Dey Chinmoy S. , Brokaw Charles J.
Published: April 2021

A high molecular mass dynein ATPase polypeptide and a 18-20 kDa dynein light chain of Ciona sperm flagella are phosphorylated during in vivo activation of motility or in vitro activation of motility by incubation with cyclic AMP. A similar level of phosphorylation of these proteins is obtained by incubation of washed, demembranated spermatozoa with catalytic subunit of cyclic AMP-dependent protein kinase, under conditions where there is no activation of motility until a supernatant component is added. Therefore, phosphorylation of these dynein polypeptides is not sufficient for activation of motility. Activation of motility in vitro by incubation with cyclic AMP can be completely inhibited by a random copolymer of glutamate and tyrosine that inhibits tyrosine kinase activity. Under these conditions, much of the protein phosphorylation associated with activation of motility is also inhibited. These new results suggest that regulation of motility of these spermatozoa may involve a multicomponent kinase cascade rather than a simple phosphorylation of a protein ‘switch’ by the cyclic AMP-dependent kinase. A 53 kDa axonemal phosphoprotein band, identified as band Ml, shows the strongest correlation with activation of motility in these experiments.
Transfection of the human muc 1 mucin gene into a poorly differentiated human pancreatic tumor cell line, pancl: Integration, expression and ultrastructural changes
Authors: Batra Surinder K. , Kern Horst F. , Worlock Andrew J. , Metzgar Richard S. , Hollingsworth Michael A.
Published: April 2021

Full-length cDNA for the human mucin Muc 1 gene under the control of the β actin promoter was transfected into a morphologically poorly differentiated pancreatic tumor cell line, Pane 1, by the DEAE-dextran method. Integration of the foreign Muc 1 cDNA occurred at multiple sites in the genome of Pane 1. Northern blot analysis showed Muc 1 expression in cells transfected with the Muc 1 cDNA, but not in control cells transfected with vector alone or an antisense Muc 1 cDNA construct. Transfection of Pane 1 with Muc 1 cDNA did not cause any detectable alteration or rearrangements in the Muc 1 gene or cDNA. Western blot analysis of cell lysates from the transfected lines using a monoclonal anti body reactive with the Muc 1 protein (HMFG-2) demonstrated that Muc 1 protein expression correlated with the Northern blot data. Immunoperoxidase staining using HMFG-2 showed that Muc 1 protein was expressed in less than 5% of control Pane 1 cells, whereas greater than 95% of cells transfected with Muc 1 cDNA expressed the protein. Ultrastructural examination of Muc 1-transfected cells demonstrated the formation of dense core granules and increased amounts of rough endoplasmic reticulum.
How individual cells develop from a syncytium: merogony in Theileria parva (Apicomplexa)
Authors: Shaw Michael K. , Tilney Lewis G.
Published: April 2021

The central problem for Theileria parva during merogony is how to form numerous individual, uninucleate merozoites from a syncytial schizont so that each merozoite contains a single nucleus and a prescribed assortment of organelles. The way T. parva packages all the requisite organelles into free merozoites is by binding these organelles to the nuclear envelope, which in turn becomes associated, both directly and through the rhoptry complex, with the schizont plasma membrane. Formation of the merozoites occurs In a synchronous manner by a budding process. The merozoites develop with the rhoptry complex at the apical end by the progressive, outward évagination of the schizont plasma membrane. This évagination of the plasma membrane is associated with, and presumably induced by, the development of an orderly array of tubules that originate from the apical end and progressively form a longitudinal basket enclosing first the rhoptry complex, then the mitochondria and ribosomes, and finally the nucleus.

The process of merogony is compared to sporogony within the tick salivary gland and with the differentiation of the intra-erythrocytic piroplasm stage. Because all three processes occur by a morphologically similar mechanism, the possibility that the parasite uses a single cassette of genes to perform each of these similar processes is discussed.
Immunolocalization of cytoplasmic dynein to lysosomes in cultured cells
Authors: Lin Sharron X. H. , Collins Christine A.
Published: April 2021

Polyclonal antisera have been raised against cytoplasmic dynein purified from calf brain and rat testis. These antibodies reacted most strongly with the 74 kDa dynein intermediate chain, but also recognized the 410 kDa heavy chain, and the 150 and 45 kDa polypeptides previously observed to copurify with cytoplasmic dynein from rat tissues. Localization studies were performed by indirect immunofluorescence microscopy using a fibroblast cell line. Dynein-specific staining appeared vesicular, distributed throughout the cell, but more concentrated near the nucleus. Double-labeling studies using fluorescent markers for membranous organelles indicated a co-localization of dynein with lysosomes. The distribution of the dynein-positive lysosomes was disrupted by treatment of the cells with microtubule-active drugs, and by acidification of the cytoplasm. Comparison of the distribution of lysosomes with peripheral microtubules indicated a high degree of coincidence. These results are consistent with the hypothesis that cytoplasmic dynein is involved in retrograde-directed movement of membranous organelles. In mitotic cells, dynein staining was also apparent along the microtubules of the mitotic apparatus, though vesicular staining was still conspicuous. The presence of dynein on vesicles as well as on spindle microtubules indicates that dynein distribution between these compartments may be regulated by distinct binding proteins.
Parental genome separation in reconstructions of somatic and premeiotic metaphases of hordeum vulgare × h. bulbosum
Authors: Schwarzacher T. , Heslop-Harrison J. S. , Anamthawat-Jónsson K. , Finch R. A. , Bennett M. D.
Published: April 2021

A stable interspecific sexual plant hybrid between Hordeum vulgare cv. Tuleen 346 (barley) × H. bulbosum was shown to have seven chromosomes originating from each parent by genomic in situ hybridization. Electron microscope serial thin-section reconstructions of metaphases and comparison with light micrograph karyotypes enabled chromosomes to be identified from their morphology. The three-dimensional positions of their centromeres were established and analysed in the reconstructions of somatic root tip metaphases and cells at mitotic metaphase near their entry into meiosis. Parental genomes tended to lie in spatially separated domains in both tissues. Although varying in morphology, the two sets of chromosomes had similar mean sizes, so size differences did not cause the separation observed. In the EM, the centromere-associated structures of the chromosomes of the more central genome, originating from H. vulgare, were larger than those of the more peripheral genome of H. bulbosum origin.
The development of a method for the preparation of rat intestinal epithelial cell primary cultures
Authors: Evans G. S. , Flint N. , Somers A. S. , Eyden B. , Potten C. S.
Published: April 2021

We describe a reproducible method for growing small intestinal epithelium (derived from the suckling rat intestine) in short-term (primary) cultures. Optimal culture conditions were determined by quantitative assays of proliferation (i.e. changes in cellularity and DNA synthesis). Isolation of the epithelia and, significantly, preservation of its three-dimensional integrity was achieved using a collagenase/dispase digestion technique. Purification of the epithelium was also facilitated by the use of a simple differential sedimentation method.

The results presented below support the idea that proliferation of normal gut epithelium ex vivo is initially dependent upon the maintenance of the structural integrity of this tissue and upon factors produced by heterologous mesenchymal cells. Proliferation in vitro was also critically dependent upon the quality of the medium and constituents used.

Cultures reached confluence within 10–14 days and consisted of epithelial colonies together with varying amounts of smooth-muscle-like cells. Cultures have been maintained for periods up to one month, but the longer-term potential for growth by sub-culturing has not been examined. Strategies for reducing the proliferation of these non-epithelial cells are also described.