bluuebunny/crossref_metadata_2025 huggingface dataset:
External alternative NADH:ubiquinone oxidoreductase redirected to the internal face of the mitochondrial inner membrane rescues complex I deficiency inYarrowia lipolytica
Authors: Kerscher Stefan J. , Eschemann Andrea , Okun Pamela M. , Brandt Ulrich
Published: April 2021

Alternative NADH:ubiquinone oxidoreductases are single subunit enzymes capable of transferring electrons from NADH to ubiquinone without contributing to the proton gradient across the respiratory membrane. The obligately aerobic yeast Yarrowia lipolytica has only one such enzyme, encoded by the NDH2 gene and located on the external face of the mitochondrial inner membrane. In sharp contrast to ndh2 deletions, deficiencies in nuclear genes for central subunits of proton pumping NADH:ubiquinone oxidoreductases (complex I) are lethal. We have redirected NDH2 to the internal face of the mitochondrial inner membrane by N-terminally attaching the mitochondrial targeting sequence of NUAM, the largest subunit of complex I. Lethality of complex I mutations was rescued by the internal, but not the external version of alternative NADH:ubiquinone oxidoreductase. Internal NDH2 also permitted growth in the presence of complex I inhibitors such as 2-decyl-4-quinazolinyl amine (DQA). Functional expression of NDH2 on both sides of the mitochondrial inner membrane indicates that alternative NADH:ubiquinone oxidoreductase requires no additional components for catalytic activity. Our findings also demonstrate that shuttle mechanisms for the transfer of redox equivalents from the matrix to the cytosolic side of the mitochondrial inner membrane are insufficient in Y. lipolytica.
The Ste5p scaffold
Authors: Elion Elaine A.
Published: April 2021

An emerging theme of mitogen-activated protein kinase (MAPK) cascades is that they form molecular assemblies within cells; the spatial organization of which is provided by scaffold proteins. Yeast Ste5p was the first MAPK cascade scaffold to be described. Early work demonstrated that Ste5p selectively tethers the MAPKKK, MAPKK and MAPK of the yeast mating pathway and is essential for efficient activation of the MAPK by the pheromone stimulus. Recent work indicates that Ste5p is not a passive scaffold but plays a direct role in the activation of the MAPKKK by a heterotrimeric G protein and PAK-type kinase. This activation event requires the formation of an active Ste5p oligomer and proper recruitment of Ste5p to a Gβγ dimer at the submembrane of the cell cortex, which suggests that Ste5p forms a stable Ste5p signalosome linked to a G protein. Additional studies underscore the importance of regulated localization of Ste5p to the plasma membrane and have revealed nuclear shuttling as a regulatory device that controls the access of Ste5p to the plasma membrane. A model that links Ste5p oligomerization with stable membrane recruitment is presented. In this model, pathway activation is coordinated with the conversion of a less active closed form of Ste5 containing a protected RING-H2 domain into an active Ste5p dimer that can bind to Gβγ and form a multimeric scaffold lattice upon which the MAPK cascade can assemble.
Role of lipid rafts in Shiga toxin 1 interaction with the apical surface of Caco-2 cells
Authors: Kovbasnjuk Olga , Edidin Michael , Donowitz Mark
Published: April 2021

Enterohemorrhagic Escherichia coli producing Shiga toxins 1 and/or 2 have become major foodborne pathogens. The specific binding of Shiga toxin 1 B-subunit to its receptor, a neutral glycolipid globotriaosylceramide Gb3, on the apical surface of colonic epithelium followed by toxin entry into cells are the initial steps of the process, which can result in toxin transcytosis and systemic effects of infection including hemolytic uremic syndrome. Understanding the complex mechanisms of Shiga toxin 1 binding and internalization may help to develop new strategies directed at preventing toxin internalization. Fluorescence resonance energy transfer microscopy revealed the clustering of Shiga toxin receptors Gb3 in lipid rafts with another glycosphingolipid GM1 on the apical surface of highly polarized intestinal epithelial Caco-2 cells. Lipid rafts disruption significantly decreased internalization of Shiga toxin 1 B-subunit. Although disruption of lipid rafts by cholesterol depletion did not affect the amount of bound Shiga toxin 1 B-subunit, lipid rafts are necessary for toxin uptake across the apical membrane of Caco-2 cells.
Potential role for laminin 5 in hypoxia-mediated apoptosis of human corneal epithelial cells
Authors: Esco Miechia A. , Wang Zhiyu , McDermott Mark L. , Kurpakus-Wheater Michelle
Published: April 2021

Laminin 5 functions to promote cell-matrix adhesion and therefore is hypothesized to abrogate apoptosis initiated through the loss of epithelial cell contact with extracellular matrix. Laminin 5 levels are decreased in epithelial cells cultured in a hypoxic environment. Exposure of epithelial cells to hypoxia may induce apoptotic pathways transmitted through changes in mitochondrial membrane potential. Using an apoptosis assay based on mitochondrial membrane integrity, the effect of hypoxia (2% oxygen) on human corneal epithelial cell viability was determined. Both a virally transformed corneal epithelial cell line and third passage corneal epithelial cells were resistant to hypoxia-mediated apoptosis for up to 5 days in culture. However, at 7 days in culture, a statistically significant increase in apoptosis was noted in hypoxic corneal epithelial cells compared to normoxic (20% oxygen) controls. Increased apoptosis in hypoxic epithelium at 7 days in culture correlated with decreased deposition of laminin 5 into the extracellular matrix, as determined by western blot analysis and immunofluorescence microscopy. Additionally, the extracellular processing of the α3 and γ2 chains of laminin 5 was negatively impacted by corneal epithelial cell exposure to hypoxia for 7 days. Treatment of human corneal epithelial cells cultured in 20% oxygen with function-inhibiting antibodies to laminin 5 for 2 or 3 days resulted in a statistically significant decrease in proliferation, and concomitant increase in apoptosis, compared with untreated normoxic controls. Based on these results, it appears that mechanisms of hypoxia-mediated apoptosis in human corneal epithelial cells may be initiated by the loss of processed laminin 5 in the extracellular matrix or by the loss of laminin 5-epithelial cell communication and transmitted through mitochondria.
Rab22a affects the morphology and function of the endocytic pathway
Authors: Mesa Rosana , Salomón Cristina , Roggero Marcelo , Stahl Philip D. , Mayorga Luis S.
Published: April 2021

Soon after endocytosis, internalized material is sorted along different pathways in a process that requires the coordinated activity of several Rab proteins. Although abundant information is available about the subcellular distribution and function of some of the endocytosis-specific Rabs (e.g. Rab5 and Rab4), very little is known about some other members of this family of proteins. To unveil some of the properties of Rab22a, one of the less studied endosome-associated small GTPases, we have expressed the protein tagged with the green fluorescent protein in CHO cells. The results indicate that Rab22a associates with early and late endosomes (labeled by a 5 minute rhodamine-transferrin uptake and the cation-independent mannose 6-phosphate receptor, respectively) but not with lysosomes (labeled by 1 hour rhodamine horseradish peroxidase uptake followed by 1 hour chase). Overexpression of the protein causes a prominent morphological enlargement of the early and late endosomes. Two mutants were generated by site-directed mutagenesis, a negative mutant (Rab22aS19N, with reduced affinity for GTP) and a constitutively active mutant (Rab22aQ64L, with reduced endogenous GTPase activity). The distribution of the negative mutant was mostly cytosolic, whereas the positive mutant associated with early and late endosomes and, interestingly also with lysosomes and autophagosomes (labeled with monodansylcadaverine). Cells expressing Rab22a wild type and Rab22aS19N displayed decreased endocytosis of a fluid phase marker. Conversely, overexpression of Rab22aQ64L, which strongly affects the morphology of endosomes, did not inhibit bulk endocytosis. Our results show that Rab22a has a unique distribution along the endocytic pathway that is not shared by any other Rab protein, and that it strongly affects the morphology and function of endosomes.
Cell fusion experiments reveal distinctly different association characteristics of cell-surface receptors
Authors: Nagy Péter , Mátyus László , Jenei Attila , Panyi György , Varga Sándor , Matkó János , Szöllősi János , Gáspár Rezső , Jovin Thomas M. , Damjanovich Sándor
Published: April 2021

The existence of small- and large-scale membrane protein clusters, containing dimers, oligomers and hundreds of proteins, respectively, has become widely accepted. However, it is largely unknown whether the internal structure of these formations is dynamic or static. Cell fusion was used to perturb the distribution of existing membrane protein clusters, and to investigate their mobility and associations. Scanning near-field optical microscopy, confocal and electron microscopy were applied to detect the exchange of proteins between large-scale protein clusters, whereas photobleaching fluorescence energy transfer was used to image the redistribution of existing small-scale membrane protein clusters. Large-scale clusters of major histocompatibility complex (MHC)-I exchanged proteins with each other and with MHC-II clusters. Similarly to MHC-I, large-scale MHC-II clusters were also dynamic. Exchange of components between small-scale protein clusters was not universal: intermixing did not take place in the case of MHC-II homoclusters; however, it was observed for homoclusters of MHC-I and for heteroclusters of MHC-I and MHC-II. These processes required a fluid state of the plasma membrane, and did not depend on endocytosis-mediated recycling of proteins. The redistribution of large-scale MHC-I clusters precedes the intermixing of small-scale clusters of MHC-I indicating a hierarchy in protein association. Investigation of a set of other proteins (α subunit of the interleukin 2 receptor, CD48 and transferrin receptor) suggested that a large-scale protein cluster usually exchanges components with the same type of clusters. These results offer new insight into processes requiring time-dependent changes in membrane protein interactions.
MTOC formation during mitotic exit in fission yeast
Authors: Heitz Molly J. , Petersen Janni , Valovin Sarah , Hagan Iain M.
Published: April 2021

Microtubules polymerise from nucleation templates containing γ tubulin. These templates are generally concentrated in discrete structures called microtubule organising centres (MTOCs). In Schizosaccharomyces pombe, an equatorial MTOC (EMTOC) forms mid-way through anaphase B and then disassembles during the final stages of cell separation. We show that the EMTOC was generated by recruiting γ tubulin to the equatorial F-actin ring before it constricted to cleave the cell in two during cytokinesis. The EMTOC was not a continuous ring. It had a variable structure ranging from a horseshoe to a number of short bars. EMTOC integrity depended upon the integrity of the F-actin but not the microtubule cytoskeleton. EMTOC assembly required the activity of both the septation-inducing network (SIN) that regulates the onset of cytokinesis and the anaphase-promoting complex. Activation of the SIN in interphase cells induced F-actin ring formation and contraction and the synthesis of the primary septum but did not promote EMTOC assembly. In contrast, overproduction of the polo-like kinase, Plo1, which also induced multiple rounds of septation in interphase cells, induced EMTOC formation. Thus, the network governing EMTOC formation shared many of the regulatory elements that control cytokinesis but was more complex and revealed an additional function for Plo1 during mitotic exit.
Distinct functional domains in emerin bind lamin A and DNA-bridging protein BAF
Authors: Lee Kenneth K. , Haraguchi Tokuko , Lee Richard S. , Koujin Takako , Hiraoka Yasushi , Wilson Katherine L.
Published: April 2021

Loss of emerin, a lamin-binding nuclear membrane protein, causes Emery-Dreifuss muscular dystrophy. We analyzed 13 site-directed mutations, and four disease-causing mutations that do not disrupt emerin stability or localization. We show that emerin binds directly to barrier-to-autointegration factor (BAF), a DNA-bridging protein, and that this binding to BAF requires conserved residues in the LEM-motif of emerin. Emerin has two distinct functional domains: the LEM-domain at the N-terminus, which mediates binding to BAF, and a second functional domain in the central region, which mediates binding to lamin A. Disease mutation Δ95-99 mapped to the lamin-binding domain and disrupted lamin A binding in vitro. Two other disease-linked residues, Ser54 and Pro183, mapped outside the BAF and lamin-binding domains, suggesting that emerin may have additional functional domains relevant to disease. The disease-linked emerin proteins all remained active for binding to BAF, both in vitro and in vivo, suggesting that disease can result from the loss of specific molecular interactions between emerin and either lamin A or putative novel partner(s). The demonstration that emerin binds directly to BAF, coupled to similar results for LAP2, provides proof in principle that all LEM-domain nuclear proteins can interact with BAF, with interesting implications for chromatin attachment to the nuclear envelope.
Fibulin-1 suppression of fibronectin-regulated cell adhesion and motility
Authors: Twal Waleed O. , Czirok Andras , Hegedus Balazs , Knaak Christian , Chintalapudi Mastan R. , Okagawa Hiroto , Sugi Yukiko , Argraves W. Scott
Published: April 2021

Fibulin-1 is an extracellular matrix protein often associated with fibronectin (FN) in vivo. In this study, the ability of fibulin-1 to modulate adhesion, spreading and motility-promoting activities of FN was investigated. Fibulin-1 was found to have pronounced inhibitory effects on the cell attachment and spreading promoted by FN. Fibulin-1 was also found to inhibit the motility of a variety of cell types on FN substrata. For example, the FN-dependent haptotactic motility of breast carcinoma (MDA MB231) cells, epidermal carcinoma (A431), melanoma (A375 SM), rat pulmonary aortic smooth muscle cells (PAC1) and Chinese hamster ovary (CHO) cells was inhibited by the presence of fibulin-1 bound to FN-coated Boyden chamber membranes. Cells transfected to overproduce fibulin-1 displayed reduced velocity, distance of movement and persistence time on FN substrata. Similarly, the incorporation of fibulin-1 into FN-containing type I collagen gels inhibited the invasion of endocardial cushion mesenchymal cells migrating from cultured embryonic heart explants. By contrast, incorporation of fibulin-1 into collagen gels lacking FN had no effect on the migration of endocardial cushion cells.

These results suggest that the motility-suppressive effects of fibulin-1 might be FN specific. Furthermore, such effects are cell-type specific, in that the migration of gingival fibroblasts and endothelial cells on FN substrata is not responsive to fibulin-1. Additional studies found that the mechanism for the motility-suppressive effects of fibulin-1 does not involve perturbations of interactions between α5β1 or α4 integrins, or heparan sulfate proteoglycans with FN. However, fibulin-1 was found to inhibit extracellular signal regulated kinase (ERK) activation and to suppress phosphorylation of myosin heavy chain. This ability to influence signal transduction cascades that modulate the actin-myosin motor complex might be the basis for the effects of fibulin-1 on adhesion and motility.
Changes in some enzymes involved in dna biosynthesis following induction of division in cultured plant cells
Authors: Harland Jennifer , Jackson J. F. , Yeoman M. M.
Published: April 2021

The biosynthetic enzymes DNA polymerase, thymidine kinase (TdR kinase) and thymidine monophosphate kinase (dTMP kinase) show characteristic patterns of activity associated with cell division in synchronously dividing tissue cultures of the Jerusalem artichoke. In each case the first major increase in enzyme activity is coincident with and dependent upon DNA replication. In addition the increased activities of all 3 enzymes are not due to the activation of pre-existing enzyme or to the removal of inhibitors. A theory is advanced to account for the close correlation between DNA synthesis and the major rise in activity of the 3 enzymes.
Visna virus infection of sheep and human cells in vitro-an ultrastructural study
Authors: Macintyre E. H. , Wintersgill C. J. , Vatter A. E.
Published: April 2021

Comparison of human astrocytes (1181N1 line) and sheep choroid plexus cells (SCP) infected with visna virus (V-1181N1 and V-SCP) shows that the mode of virus assembly and the morphology and size of the free virions are identical in the 2 systems. V-SCP cells usually cytolyse soon after infection; V-1181N1 cells do not. This is considered to be due to inter ference with membrane assembly in the former. Syncytial giant cells from both V-1181N1 and V-SCP cultures have a much looser attachment to the growth surface than their un infected parents. This is shown to be associated with a loss of certain microfibrillar arrays close to the plasmalemma. Uninfected SCP cultures have cilia and desmosomes and retain these after infection till cytolysis; 1181N1 cells do not have these structures, but have a well developed microtubular system, which the SCP cells lack.
Histochemical and biochemical demonstration of sialic acid and sulphate in vesicles and membranes isolated from nerve endings of rat brain
Authors: Marx Ruth , Graf Elke , Wesemann W.
Published: April 2021

The reaction of colloidal iron hydroxide (CIH) with acidic groups was applied for an ultra structural study of the distribution of sulphuric acid monoesters and sialic acid in synaptic vesicles and external nerve ending membranes isolated from rat brain. At pH 1·7 CIH was precipitated as electron-dense granules with a uniform size of 6—7 nm specifically labelling the carboxyl group of sialic acid and the sulphate group of monoesters of sulphuric acid. The differentiation of these 2 groups was achieved by treatment with neuraminidase and methylation followed by saponification. After preincubation with neuraminidase, which released 90—100 % of the sialic acid from the membranes of the synaptic vesicles and the nerve endings, the electron-dense deposits marked the reaction sites of sulphate with CIH. The sulphate groups which were present at a concentration of 2·3 and 2·2 μmol/mg protein for the synaptic vesicle and nerve ending membrane preparations, respectively, were rendered soluble as methyl monosulphate by trans-esterification with acid/methanol and quantitatively removed from the structures. By this treatment membrane-bound sialic acid was blocked as sialic acid methyl ester and partly split off by acid hydrolysis. About 55 % of the sialic acid found in the nerve ending membranes remained attached to the structure as compared with about 35 % of the sialic acid of the vesicles. The acid-resistant proportion of the sialic acid could be localized with CIH after saponification of the esterified preparations. The method described allows the electron-microscopical demonstration of acid-resistant, neuraminidase-sensitive sialic acid in synaptic structures and the discrimination from sulphated mucopolysaccharides.
Structural and functional modifications of the nucleus during oogenesis in the mosquito Aedes aegypti
Authors: Fiil Annelise
Published: April 2021

The observations are based on reconstructions from electron micrographs of complete serial sections of some 20 oocytes at different stages of development. At eclosion the Aedes aegypti egg chambers each contain 8 interconnected cells, one oocyte and 7 nurse cells. During the next few days the egg chambers develop up to a previtellogenic resting stage. The most advanced oocytes are now in the diplotene stage of meiosis. Following the first blood meal vitellogenesis commences and lasts for 3−4 days. During this time the oocyte increases from a sphere with a diameter of 30μm to an elongate spheroid of the dimensions 190 × 600μm. The nucleus expands with the oocyte, first branching then forming a cup-shaped structure, and finally elongating to a large sheet-like, cylindrical form. The chromosomes, condensed into a karyosphere, remain at the anterior pole close to the nurse cells. Towards the end of vitellogenesis meiosis is resumed, and shortly thereafter the envelope of the large nucleus disintegrates. For a short period the chromosomes, which are in first meiotic metaphase, are without an enveloping membrane, then several layers of vesiculate membrane reassemble around them, trapping some of the cytoplasm in the process. At this stage the oocyte may be ovulated or it may rest unchanged for some time. Thirty minutes after oviposition meiosis is complete, and the masses of membranes assemble in a few regions which later degenerate with the polar bodies.
Applications of a theory of biological pattern formation based on lateral inhibition
Authors: Meinhardt H. , Gierer A.
Published: April 2021

Model calculations are presented for various problems of development on the basis of a theory of primary pattern formation which we previously proposed. The theory involves shortrange autocatalytic activation and longer-range inhibition (lateral inhibition). When a certain criterion is satisfied, self-regulating patterns are generated.

The autocatalytic features of the theory are demonstrated by simulations of the determination of polarity in the Xenopus retina. General conditions for marginal and internal activation, and corresponding effects of symmetry are discussed. Special molecular mechanisms of pattern formation are proposed in which activator is chemically converted into inhibitor, or an activator precursor is depleted by conversion into activator.

The (slow) effects of primary patterns on differentiation can be included into the formalism in a straightforward manner. In conjunction with growth, this can lead to asymmetric steady states of cell types, cell differentiation and proliferation as found, for instance, in growing and budding hydra. In 2 dimensions, 2 different types of patterns can be obtained. Under some assumptions, a single pattern-forming system produces a ‘bristle’ type pattern of peaks of activity with rather regular spacings on a surface. Budding of hydra is treated on this basis. If, however, gradients’ develop under the influence of a weak external or marginal asymmetry, a monotonic gradient can be formed across the entire field, and 2 such gradient-forming systems can specify ‘positional information’ in 2 dimensions.

If inhibitor equilibrates slowly, a spatial pattern may oscillate, as observed with regard to the intracellular activation of cellular slime moulds.

The applications are intended to demonstrate the ability of the proposed theory to explain properties frequently encountered in developing systems.
Sulphur in different types of keratohyalin granules: a quantitative assay by x-ray microanalysis
Authors: Jessen H. , Peters P. D. , Hall T. A.
Published: April 2021

The elemental composition of keratohyalin granules from the interpapillary and papillary lingual epithelium and the oesophageal epithelium of the rat were studied by X-ray microanalysis in an EMMA-4 analytical electton microscope, equipped with an energy dispersive detector. A quantitative assay of the sulphur concentration in keratohyalin granules was performed, using a suitable sulphur standard. The results demonstrate that different types of keratohyalin granule have different compositions. Single granules - a type of keratohyalin granule present in both nuclei and cytoplasm of the epithelial cells - are rich in sulphur, having a content of 3·6-3·8%. Another type of keratohyalin granule - composite granules - contains a sulphur-rich component and a sulphur-poor component. The sulphur-poor component contains 0··8-1·4% sulphur. It is suggested that the sulphur-rich keratohyalin granules are involved in the deposition of the peripheral envelope protein of cornified cells.
Cytochalasin b: aspects of phagocytosis in nutrient uptake in tetrahymena
Authors: Hoffmann Else K. , Rasmussen L. , Zeuthen E.
Published: April 2021

Cytochalasin B (37 μg per ml) reduces the rate of food vacuole formation, i.e. the rate of phagocytosis, in Tetrahymena pyriformis. Cytochalasin B in this concentration suppresses multiplication rates in a nutrient medium consisting of 2 % proteose peptone, but multiplication is unaffected if this medium is supplemented with glucose and high concentrations of nucleosides. Thus nutrients in high concentrations circumvent the necessity for phagocytosis in Tetrahymena.
Contractile filopodia and in vivo cell movement in the tunic of the ascidian, Botryllus schlosseri
Authors: Izzard C. S.
Published: April 2021

The in vivo movement of one class of cells in the tunic of the ascidian Botryllus schlosseri has been analysed using differential interference optics and time-lapse cinematography. Long (up to 200 μm), thin (0·35–0·5 pm diameter) filopodia radiate from the cell-body into the matrix of the tunic. Movement of the cell-body consists of a series of short, jerky displacements with frequent changes in direction between successive displacements. The net displacement of the cell may be extremely small when the displacements are short and frequently change direction, or considerable when successive displacements show a persistence of direction (up to 114 μm in 60 min). Deformation of the elastic cuticle covering the tunic at points of attachment of the filopodia has been used to record qualitatively changes in tension in the filopodia. Correlation of the changes in tension with changes in length of the filopodia and movement of the cell-body have permitted the following conclusions. Active contractions of filopodia (i.e. increase in tension during shortening) stretch and move the cell-body. These movements exert a force on trailing or opposing filopodia. Relaxations of filopodia (i.e. decrease in tension during lengthening) result in small movements of the cell-body due to the recoil of tension in the cell-body and opposing filopodia. The position of the cell-body in space at any one instant in time is therefore the resultant of the forces developed in all the filopodia. Movement results from unilateral modulation of the tension developed in the filopodia. Active contractions play a more significant role in movement than relaxations.
The control of karyogamy in somatic cells of Ustilago violacea
Authors: Day A. W. , Day Lynne L.
Published: April 2021

The rate of nuclear fusion (karyogamy) in the haploid smut fungus, Ustilago violacea, designated D, can be determined accurately from the frequency of diploid formation. The spontaneous value of D depends mainly on the age of the cells and the temperature, varying from 10−5 to 10−2. Ultraviolet light (u.v., 254 nm) induces high rates of fusion, up to too % of the survivors being diploid after very high doses. That this increase is caused by induction and not by selection is shown by the absolute increase in the number of diploids obtained under some conditions. The high value of D after a u.v. treatment is reduced by conditions which favour photoreactivation or dark repair. Acriflavine, an inhibitor of dark repair, maintains high values of D, while caffeine which also inhibits dark repair abolishes the u.v. induction entirely.

Ultraviolet-sensitive mutants located at 4 loci were tested for their effect on spontaneous and u.v.-induced karyogamy. One mutant (uvs4) showed complete absence of somatic karyogamy even after high doses of ultraviolet. Mutant uvs2 had no effect on the value of D, while mutants uvs1 and uvs3 showed low spontaneous karyogamy, but could be induced by low doses of u.v. to yield large numbers of diploids.

It is suggested that an inhibitor of karyogamy is formed in somatic cells and destroyed prior to premeiotic karyogamy. The accuracy of the transcription of this inhibitor seems to be impaired by ultraviolet, but can be restored following DNA repair. The absence of somatic karyogamy in uvs4 mutants may indicate that these strains contain an altered organelle involved in the control of karyogamy, e.g. the spindle pole body situated on the nuclear membrane, and known to contain DNA. Alteration of such an organelle could produce pleiotropic effects including u.v. sensitivity and inhibitor production or action.
The localization of calcium and phosphorus in human platelets
Authors: Skaer R. J. , Peters P. D. , Emmines J. P.
Published: April 2021

Large amounts of calcium and phosphorus have been found by electron-microprobe analysis in the dense bodies of frozen-dried, human platelets. The calcium and phosphorus occurred in a fixed ratio that was similar to that in dicalcium ATP. No other elements were detectable except chlorine. Minute, electron-dense disks were found in the surface membranes of glutaraldehyde-fixed platelets, and are very variable in number in any population of normal platelets. Both dense bodies and membrane granules are absent if platelets are fixed in the absence of calcium, and are also absent from aggregated platelets.
Decrease in section thickness on exposure to the electron beam; the use of tilted sections in estimating the amount of shrinkage
Authors: Bennett Pauline M.
Published: April 2021

Tilting experiments on sections of embedded light meromyosin paracrystals of known periodic structure indicate that the section thickness decreases during examination in the electron microscope. The material remaining in the section after irradiation collapses uniformly to about one half the initial thickness. This must be taken into account when measurements are made on tilted sections.
Visualization of charged groups on the surface of rat liver nuclei
Authors: Virtanen Ismo , Wartiovaara Jorma
Published: April 2021

Anionic groups on the outer surfaces of isolated rat liver nuclei were rendered visible in the electron microscope by staining with colloidal iron hydroxide at different pH values. At pH 1·8 the nuclei did not adsorb particles of stain, although plasma membranes left in the same preparation showed heavy labelling. After pretreatment with neuraminidase at pH 6 the plasma membranes were no longer stained. At pH 3·0 the nuclear surfaces also stained intensely. The staining pattern acquired at this pH did not appear to be changed by neuraminidase pretreatment.

With the staining method used, rat liver nuclear surfaces seemed to have no exposed sialic acid under isolation conditions which preserve the nuclear membranes and leave the ribosomes attached to the nuclear surface. However, at higher pH values other anionic groups seem to become dissociated and are stained with colloidal iron hydroxide.
Observations on the male nucleus during fertilization in the fern Pteridium aquilinum
Authors: Bell P. R.
Published: April 2021

Fertilization of eggs of Pteridium under controlled conditions has revealed that archegonia containing viable eggs require more than 30 min flooding before opening. Syngamy appears to take place by an engulfing action, and no evidence was found of a ‘membrane-fusion’ kind of conjugation.

Although the gametic nucleus lacks a recognizable envelope, as soon as it comes into contact with the female cytoplasm an envelope is reconstituted from an opaque layer at the surface of its chromatin. This membrane also undergoes growth, the outer layer extending into the cytoplasm and surrounding spermatozoid mitochondria. The male envelope remains distinct from the female, and lacks evaginations and well defined pores.

The male nucleus lies in irregular loops within a funnel, filled with egg cytoplasm, formed by the microtubular ribbon of the gamete. The mouth of the funnel is bordered by the multilayered structure. Karyogamy is initiated at the anterior end of the male nucleus, the orifice connecting the 2 nuclei widening from about 0·7 to 2 μm in diameter during fusion. The contents of the male nucleus flow through the mouth of the funnel into the female, the chromatin relaxing locally, but retaining a rod-like form. The envelope of the male nucleus is probably resorbed at the site of junction. There is no evidence that it contributes significantly to the envelope of the zygotic nucleus.
A light- and electron-microscope study of the oocyte nucleus during development of the antral follicle in the prepubertal mouse
Authors: Chouinard L. A.
Published: April 2021

The ordered changes which occur in the structural organization of the mouse oocyte nucleus during the preparatory, the maturative and the preovulatory stages of antral follicle development, have been studied under both light and electron microscopy. All observations have been made on those antral follicles whose development is initiated on postnatal day 14 and completed by postnatal day 28 in prepubertal animals of the ICR albino mouse strain. The formed entities that can be recognized within the oocyte nucleus during that period are the condensing bivalents, the heterochromatic knobs, the nucleolus and the extranucleolar bodies. At the onset of antral follicle development, the highly unravelled dictyate bivalents are seen to take on a lampbrush-type configuration. Subsequent condensation of these lampbrush bivalents appears to be a very gradual and lengthy process that extends over almost the entire period of antral follicle development. The shortening and thickening of the lampbrush bivalents are best interpreted as resulting from the withdrawal of their lateral loop-like projections into the chromosome axes and from the focal aggregation of these axes into compact chromatin masses. Electron-opaque granules, which appear within the oocyte nucleus during the preparatory and maturative follicle stages, are seen to be intimately associated with these condensing bivalents. A number of Feulgen-positive heterochromatic knobs make their appearance in contact with certain bivalents during the preparatory follicle stage. These knobs are not reincorporated as such into the condensing chromatin masses and undergo disintegration and dissolution during the preovulatory follicle stage. The size, shape and ultrastructural features of the nucleolus remain unchanged throughout the period of antral follicle development. Breakdown and dissolution of the nucleolar mass is a swift process that takes place only in the fully mature preovulatory follicle and more or less concomitantly with the dismantling of the nuclear envelope. The extranucleolar bodies increase noticeably in size during the preparatory and the maturative follicle stages; they shrink in size and undergo dissolution during the preovulatory stage of antral follicle development.

An attempt is made to interpret these morphological changes in the light of current knowledge concerning the architectural and functional organization of the oocyte nucleus in general during meiotic prophase. The relevant observational evidence would be consistent with the view that, during antral follicle development, the mouse oocyte nucleus is not, as too often assumed, in a period of arrested evolution; its formed components undergo structural, maturational and functional changes which are of significance not only for the resumption of the first meiotic prophase but also for the early development of the embryo.
The binding of the mucoprotein from gastric mucus to cells in tissue culture and the inhibition of cell adhesion
Authors: Allen A. , Minnikin S.Megan
Published: April 2021

The mucoprotein, which is responsible for the formation of gastric mucous gel in pig, has been shown to bind equally well to suspensions of baby hamster kidney cells, polyoma-virus-transformed baby hamster kidney cells and HeLa cells. The binding of the mucoprotein to the cells is dependent on Ca2+/Mg2+ ions and is 5-fold greater at 37 than 4 °C. Prolonged treatment of the cells with trypsin reduces their binding capacity for the mucoprotein to less than half. The rate of adhesion of single “S-labelled baby hamster kidney cells, polyoma-transformed baby hamster kidney cells and HeLa cells to the different monolayers has been studied and found to be about the same for all 3 types of cell. The same gastric mucoprotein which bound to the cells also inhibited the adhesion of the different types of single cells to the monolayers. Thus, about one μmol l.-1 of gastric mucoprotein reduced the adhesion of single BHK cells to BHK monolayers to one-seventh of that in the absence of mucoprotein. The binding of the mucoprotein to the cells is essentially irreversible, and also single cells preincubated with the mucoprotein do not adhere to the monolayers when no free mucoprotein is present. Both the binding of the mucoprotein to the cells and its inhibitory action on cell adhesion are abolished if high concentrations of calf serum are included in the medium. Red blood cells neither adhere to each other nor do they bind the mucoprotein. The interaction of the mucoprotein with the cells is specific in that bovine serum albumin, calf serum, glycogen and hyaluronic acid neither affected the adhesion between the cells nor in the case of bovine serum albumin bound significantly. Furthermore, measurements of binding show that only a fraction of the purified mucoprotein (6%) has the necessary primary and/or tertiary structure to combine with the receptor sites on the cell surface. The similarities between the adhesion of the cells to monolayers and the binding of the gastric mucoprotein to these cells is discussed in terms of the importance of cell surface mucoproteins in cell adhesion.
Fine structure of the haplosporidan Kernstab, a persistent, intranuclear mitotic apparatus
Authors: Perkins F. O.
Published: April 2021

The fine structure of the haplosporidan mitotic apparatus is described from observations of plasmodial nuclei of Minchinia nelsoni, M. costalis, Minchinia sp., and Urosporidium crescens. The apparatus, which is the Kernstab of light-microscope studies, consists of a bundle of microtubules terminating in a spindle pole body (SPB) at each end of the bundle. A few microtubules extend from SPB to SPB, but most either extend from an SPB and terminate in the nucleoplasm or lie in the nucleoplasm, free of either SPB. The bundle lengthens during mitosis, increasing the SPB-to-SPB distance by a factor of 2 to 3 as compared to interphase nuclei. SPBs arc not in contact with the nuclear envelope, being found always in the nucleoplasm which is delimited by the nuclear envelope throughout mitosis. The mitotic apparatus is persistent through interphase, at least in a form which is not significantly different from that found in mitotic nuclei.
Cell locomotion within a contact-inhibited monolayer of chick embryonic liver parenchyma cells
Authors: Garrod D. R. , Steinberg M. S.
Published: April 2021

Using time-lapse filming, the relative movement of cells (nuclei) within a contact-inhibited monolayer of chick embryonic liver parenchyma cells has been studied. Two techniques were employed to determine the amount of relative cell movement during a culture period of 6 h. Firstly, the number of neighbours lost or gained by each nucleus was counted. Secondly, the relative distance moved by each nucleus in relation to other nuclei in the monolayer was measured. (The numerical results obtained from these analyses and details of the methods used are given in the text.) A considerable amount of relative movement of nuclei within the monolayer was found during this short period of culture.

Although some gaps were occasionally seen between the cells in the monolayer, it was observed that cells were able both to ‘ruffle ‘and to translocate when no gap was detectable; i.e. the cells appeared able to move while entirely surrounded by other cells. Because of this, we suggest that the monolayering of these epithelioid cells on a surface may be due to restriction of overlapping between them rather than to inhibition of movement by mutual contact. We argue that the term ‘contact inhibition of overlapping’ relates to this behaviour better than the term ‘contact inhibition of movement’.
The erythroid cells of anaemic Xenopus laevis. II. Studies on nuclear non-histone proteins
Authors: Hilder V. A. , Thomas Nesta , Maclean N.
Published: April 2021

The mass ratio of nuclear non-histone protein: DNA in the immature circulating erythroid cells of phenylhydrazine-induced anaemic Xenopus is approximately threefold higher than in mature erythrocytes. This is largely due to the presence of increased amounts of low and intermediate molecular weight proteins in the nuclei of the immature cells. There are a few qualitative differences in the components of this class of proteins between the mature and immature cells, the most notable of which is the presence of a protein of molecular weight approximately 115 000 in the former which is not detectable in the latter. These changes are discussed in relation to the changing synthetic capacities of the cells and to certain generalizations about the function of the nuclear non-histone proteins based on studies of other differentiating systems.
The light microscope as an optical diffractometer
Authors: Gall J. G.
Published: April 2021

The analysis of electron micrographs by optical diffraction was introduced recently by Klug & Berger (1964). Their experiments were conducted with a special diffractometer designed for use with diffracting masks up to several inches in diameter. A method is described here for using a conventional light microscope as an optical diffractometer which can accept masks up to 5 mm in diameter. A-100 μ electron-microscope aperture is used as a pinhole source of illumination, and the micrograph to be studied is introduced above the objective. The diffraction pattern produced by the micrograph appears in the usual image plane of the microscope within the eyepiece.
Identification of glycogen in thin sections of amphibian embryos
Authors: Perry Margaret M.
Published: April 2021

Embryonic amphibian cells when examined with the electron microscope were observed to contain an abundance of small particles, approximately 325 Å in diameter. The periodic acid/ Schiff reaction and enzymic digestion were employed to determine the nature of the particles, and from the results of these tests they were concluded to be glycogen. Treatment of thin sections with periodic acid/lead citrate solutions resulted in a marked increase in contrast of the glycogen particles compared with other cell structures, and in a clearly defined substructure of 40-Å grains appearing within the particles. This differential staining method enabled the particulate glycogen to be distinguished from ribosomes.
Further observations on the fine structure of Chrysochromulina chiton with special reference to the haptonema, ‘peculiar’ golgi structure and scale production
Authors: Manton Irene
Published: April 2021

A more detailed study of ‘peculiar’ Golgi structure in this organism has been attempted than in any similar species, using modem fixation methods. Where comparisons are possible there is close resemblance to the related genus Prymnesium, to a degree sufficient to suggest that this particular type of Golgi system is likely to be a major phyletic indicator for the class Hapto-phyceae. Certain stages of scale production have been traced and some developmental aspects of the whole system are discussed, though full interpretation is deferred. Some additional features of haptonema structure are described for the sake of completeness in characterizing the particular strain of the species under investigation. These include information on the internal structure of the haptonema tip.
Fine structure of chicken thymic epithelial vesicles
Authors: Isler H.
Published: April 2021

This paper describes the fine structure of epithelial vesicles as observed in the thymus of the chicken. Three types of vesicles are found. The largest is made up of a stratified columnar epithelium containing lymphoid cells. The lumen contains a fairly light material and cell debris. The lumen-bordering cells are columnar and their microvilli are sometimes developed to such an extent as to appear as a brush border. The wall of the vesicles contains lymphocytes and granular cells with dense granules 100–500 nm in diameter. Smaller vesicles are found which contain no lymphoid cells. They are made of a simple or stratified epithelium containing typical mucous cells, and columnar cells similar to the bordering cells of the large vesicles. The third type is an intracytoplasmic vesicle in epithelial reticular cells, which differs from usual vacuoles by the presence of microvilli and its often unusually large size. The morphology of the first 2 types of vesicles indicates a secretory capability, the function of which is unknown. Data from the literature suggest that the third type produces the factor(s) conferring cellular immunological competence.
Fine-Structural Studies of the Gametes and Embryo ofFucus VesiculosusL. (Phaeophyta): I. Fertilization and Pronuclear Fusion*
Authors: Brawley Susan H. , Wetherbee Richard , Quatrano Ralph S.
Published: April 2021

In the marine brown alga, Fucus vesiculosus L., the sperm pronucleus is delimited by an envelope following penetration of the egg by the sperm. This envelope disintegrates as the pronucleus begins its migration through the cytoplasm of the egg. The highly condensed chromatin of the sperm pronucleus disperses slightly following disintegration of the envelope. Microtubules of unknown origin are associated with the sperm pronucleus during its migration. The flagellar microtubules remain in the peripheral cytoplasm but lose their tight 9 + 2 configuration. The sperm eyespot and mitochondria follow the pronucleus through the cytoplasm toward the egg pronucleus. The mitochondria of the sperm are distinguished from those of the egg by their longitudinally oriented cristae and by electron-opaque material in the intracristal space. The pronucleus of the egg becomes convoluted along the surface nearest to the advancing sperm pronucleus. Immediately prior to pronuclear fusion, many egg mitochondria aggregate in the vicinity of the sperm pronucleus. At this time, only the portion of the sperm pronucleus facing the egg pronucleus is surrounded by an envelope. The egg mitochondria disperse rapidly after pronuclear fusion. The sperm mitochondria and eyespot are still in the perinuclear region in 16-h-old embryos. At this time, the osmiophilia of the sperm eyespot has increased, and the sperm mitochondrial membranes are less distinct than in earlier stages. The fine-structural features of fertilization in Fucus are discussed in relation to the fertilization patterns in other cryptogams and marine invertebrates and to polar axis determination in the Fucaceae.
Electron Microscopy of Interphase Chromosomes In Situ: Binding of Permanganate to Chicken Erythrocytes
Authors: Davies H. G.
Published: April 2021

From quantitative electron-microscope observations on the binding of permanganate to regions of erythrocytes and reticulocytes of known chemical composition, it is concluded that KMnO4, like phosphotungstic acid (PTA), binds preferentially to sites on proteins. Compared with PTA, KMnO4 binding exhibits less anomalous behaviour. The data support the hypothesis previously put forward that the 2 regions, or phases, in condensed chromatin differ in both molecular composition and concentration. The increase in binding to protein which occurs during nuclear haemolysis is interpreted in terms of protein–protein interaction in the chromatin of the intact cell.