bluuebunny/crossref_metadata_2025 huggingface dataset:
Misfolded growth hormone causes fragmentation of the Golgi apparatus and disrupts endoplasmic reticulum-to-Golgi traffic
Authors: Graves Thomas K. , Patel Shilpa , Dannies Priscilla S. , Hinkle Patricia M.
Published: April 2021

In some individuals with autosomal dominant isolated growth hormone deficiency, one copy of growth hormone lacks amino acids 32-71 and is severely misfolded. We transfected COS7 cells with either wild-type human growth hormone or Δ32-71 growth hormone and investigated subcellular localization of growth hormone and other proteins. Δ32-71 growth hormone was retained in the endoplasmic reticulum, whereas wild-type hormone accumulated in the Golgi apparatus. When cells transfected with wild-type or Δ32-71 growth hormone were dually stained for growth hormone and the Golgi markers β-COP, membrin or 58K, wild-type growth hormone was colocalized with the Golgi markers, but β-COP, membrin and 58K immunoreactivity was highly dispersed or undetectable in cells expressing Δ32-71 growth hormone. Examination of α-tubulin immunostaining showed that the cytoplasmic microtubular arrangement was normal in cells expressing wild-type growth hormone, but microtubule-organizing centers were absent in nearly all cells expressing Δ32-71 growth hormone. To determine whether Δ32-71 growth hormone would alter trafficking of a plasma membrane protein, we cotransfected the cells with the thyrotropin-releasing hormone (TRH) receptor and either wild-type or Δ32-71 growth hormone. Cells expressing Δ32-71 growth hormone, unlike those expressing wild-type growth hormone, failed to show normal TRH receptor localization or binding. Expression of Δ32-71 growth hormone also disrupted the trafficking of two secretory proteins, prolactin and secreted alkaline phosphatase. Δ32-71 growth hormone only weakly elicited the unfolded protein response as indicated by induction of BiP mRNA. Pharmacological induction of the unfolded protein response partially prevented deletion mutant-induced Golgi fragmentation and partially restored normal TRH receptor trafficking. The ability of some misfolded proteins to block endoplasmic reticulum-to-Golgi traffic may explain their toxic effects on host cells and suggests possible strategies for therapeutic interventions.
Transient upregulation of the glial glutamate transporter GLAST in response to fibroblast growth factor, insulin-like growth factor and epidermal growth factor in cultured astrocytes
Authors: Suzuki Keiko , Ikegaya Yuji , Matsuura Sigeru , Kanai Yoshikatsu , Endou Hitoshi , Matsuki Norio
Published: April 2021

Although expression of the glial glutamate transporter GLAST is tightly regulated during development and under pathophysiological conditions, little is known about endogenous modulators of GLAST expression. Because growth factors are generally believed to regulate glial functions, we addressed their possible contribution to GLAST regulation in cultured rat astrocytes. Of the six growth factors tested (basic fibroblast growth factor (bFGF), insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), insulin, platelet-derived growth factor, and hepatocyte growth factor), bFGF, IGF-1 and EGF enhanced [3H]glutamate transport activity in a concentration-dependent manner. These effects were accompanied by an increase in the Vmax value for transport activity and in GLAST protein and mRNA levels, which suggests that GLAST expression is transcriptionally regulated by the growth factors. Interestingly, the effects reached a peak after 36 hours of exposure to growth factors, and rapidly returned to baseline by 48 hours. A combination of IGF-1 with either bFGF or EGF showed an additive effect on the glutamate uptake activity, but a combination of bFGF and EGF did not. Pharmacological blockade of protein kinase C inhibited the effects of IGF-1 and EGF, but not bFGF. By contrast, genistein, an inhibitor of tyrosine kinases, blocked the effects of bFGF and EGF without affecting the effect of IGF-1. These results suggest that the growth factors activate different signaling pathways for GLAST upregulation. The present study may indicate a novel regulatory system of glial glutamate transporters.
Gain-of-function of poly(ADP-ribose) polymerase-1 upon cleavage by apoptotic proteases: implications for apoptosis
Authors: D’Amours Damien , Sallmann Frédéric R. , Dixit Vishva M. , Poirier Guy G.
Published: April 2021

Poly(ADP-ribosyl)ation is an important mechanism for the maintenance of genomic integrity in response to DNA damage. The enzyme responsible for poly(ADP-ribose) synthesis, poly(ADP-ribose) polymerase 1 (PARP-1), has been implicated in two distinct modes of cell death induced by DNA damage, namely apoptosis and necrosis. During the execution phase of apoptosis, PARP-1 is specifically proteolyzed by caspases to produce an N-terminal DNA-binding domain (DBD) and a C-terminal catalytic fragment. The functional consequence of this proteolytic event is not known. However, it has recently been shown that overactivation of full-length PARP-1 can result in energy depletion and necrosis in dying cells. Here, we investigate the molecular basis for the differential involvement of PARP-1 in these two types of cellular demise. We show that the C-terminal apoptotic fragment of PARP-1 loses its DNA-dependent catalytic activity upon cleavage with caspase 3. However, the N-terminal apoptotic fragment, retains a strong DNA-binding activity and totally inhibits the catalytic activity of uncleaved PARP-1. This dominant-negative behavior was confirmed and extended in cellular extracts where DNA repair was completely inhibited by nanomolar concentrations of the N-terminal fragment. Furthermore, overexpression of the apoptotic DBD in mouse fibroblast inhibits endogenous PARP-1 activity very efficiently in vivo, thereby confirming our biochemical observations. Taken together, these experiments indicate that the apoptotic DBD of PARP-1 acts cooperatively with the proteolytic inactivation of the enzyme to trans-inhibit NAD hydrolysis and to maintain the energy levels of the cell. These results are consistent with a model in which cleavage of PARP-1 promotes apoptosis by preventing DNA repair-induced survival and by blocking energy depletion-induced necrosis.
Cell motility: can Rho GTPases and microtubules point the way?
Authors: Wittmann Torsten , Waterman-Storer Clare M.
Published: April 2021

Migrating cells display a characteristic polarization of the actin cytoskeleton. Actin filaments polymerise in the protruding front of the cell whereas actin filament bundles contract in the cell body, which results in retraction of the cell’s rear. The dynamic organization of the actin cytoskeleton provides the force for cell motility and is regulated by small GTPases of the Rho family, in particular Rac1, RhoA and Cdc42. Although the microtubule cytoskeleton is also polarized in a migrating cell, and microtubules are essential for the directed migration of many cell types, their role in cell motility is not well understood at a molecular level. Here, we discuss the potential molecular mechanisms for interplay of microtubules, actin and Rho GTPase signalling in cell polarization and motility. Recent evidence suggests that microtubules locally modulate the activity of Rho GTPases and, conversely, Rho GTPases might be responsible for the initial polarization of the microtubule cytoskeleton. Thus, microtubules might be part of a positive feedback mechanism that maintains the stable polarization of a directionally migrating cell.
Process formation results from the imbalance between motor-mediated forces
Authors: Ferhat Lotfi , Rami Guillaume , Medina Igor , Ben-Ari Yehezkel , Represa Alfonso
Published: April 2021

Several reports have suggested that neurite outgrowth is mediated by opposing forces generated on microtubules and microfilaments but the molecular basis underlying these forces have not been determined. Here, we show that in non-neuronal cell lines, the inhibition of actomyosin activity by acidic calponin promotes the formation of processes. This effect is blocked by inhibition of the motor activity of cytoplasmic dynein. Therefore, neurite formation is due to an imbalance between tensile and compressive forces mediated by myosins and dyneins, respectively. We propose a mechanism that involves the motor-mediated forces in a tight regulation of the process formation.
Identification, characterization and localization of chagasin, a tight-binding cysteine protease inhibitor inTrypanosoma cruzi
Authors: Monteiro Ana C. S. , Abrahamson Magnus , Lima Ana P. C. A. , Vannier-Santos Marcos A. , Scharfstein Julio
Published: April 2021

Lysosomal cysteine proteases from mammalian cells and plants are regulated by endogenous tight-binding inhibitors from the cystatin superfamily. The presence of cystatin-like inhibitors in lower eukaryotes such as protozoan parasites has not yet been demonstrated, although these cells express large quantities of cysteine proteases and may also count on endogenous inhibitors to regulate cellular proteolysis. Trypanosoma cruzi, the causative agent of Chagas’ heart disease, is a relevant model to explore this possibility because these intracellular parasites rely on their major lysosomal cysteine protease (cruzipain) to invade and multiply in mammalian host cells. Here we report the isolation, biochemical characterization, developmental stage distribution and subcellular localization of chagasin, an endogenous cysteine protease inhibitor in T. cruzi. We used high temperature induced denaturation to isolate a heat-stable cruzipain-binding protein (apparent molecular mass, 12 kDa) from epimastigote lysates. This protein was subsequently characterized as a tight-binding and reversible inhibitor of papain-like cysteine proteases. Immunoblotting indicated that the expression of chagasin is developmentally regulated and inversely correlated with that of cruzipain. Gold-labeled antibodies localized chagasin to the flagellar pocket and cytoplasmic vesicles of trypomastigotes and to the cell surface of amastigotes. Binding assays performed by probing living parasites with fluorescein (FITC)-cruzipain or FITC-chagasin revealed the presence of both inhibitor and protease at the cell surface of amastigotes. The intersection of chagasin and cruzipain trafficking pathways may represent a checkpoint for downstream regulation of proteolysis in trypanosomatid protozoa.
Calcium puffs are generic InsP3-activated elementary calcium signals and are downregulated by prolonged hormonal stimulation to inhibit cellular calcium responses
Authors: Tovey Stephen C. , de Smet Patrick , Lipp Peter , Thomas David , Young Kenneth W. , Missiaen Ludwig , De Smedt Humbert , Parys Jan B. , Berridge Michael J. , Thuring Jan , Holmes Andrew , Bootman Martin D.
Published: April 2021

Elementary Ca2+ signals, such as ‘Ca2+ puffs’, which arise from the activation of inositol 1,4,5-trisphosphate receptors, are building blocks for local and global Ca2+ signalling. We characterized Ca2+ puffs in six cell types that expressed differing ratios of the three inositol 1,4,5-trisphosphate receptor isoforms. The amplitudes, spatial spreads and kinetics of the events were similar in each of the cell types. The resemblance of Ca2+ puffs in these cell types suggests that they are a generic elementary Ca2+ signal and, furthermore, that the different inositol 1,4,5-trisphosphate isoforms are functionally redundant at the level of subcellular Ca2+ signalling. Hormonal stimulation of SH-SY5Y neuroblastoma cells and HeLa cells for several hours downregulated inositol 1,4,5-trisphosphate expression and concomitantly altered the properties of the Ca2+ puffs. The amplitude and duration of Ca2+ puffs were substantially reduced. In addition, the number of Ca2+ puff sites active during the onset of a Ca2+ wave declined. The consequence of the changes in Ca2+ puff properties was that cells displayed a lower propensity to trigger regenerative Ca2+ waves. Therefore, Ca2+ puffs underlie inositol 1,4,5-trisphosphate signalling in diverse cell types and are focal points for regulation of cellular responses.
A new HIF-1 alpha variant induced by zinc ion suppresses HIF-1-mediated hypoxic responses
Authors: Chun Yang-Sook , Choi Eunjoo , Yeo Eun-Jin , Lee Jong Ho , Kim Myung-Suk , Park Jong-Wan
Published: April 2021

The expressions of hypoxia-inducible genes are upregulated by hypoxia-inducible factor 1 (HIF-1), which is a heterodimer of HIF-1α and HIF-1β/ARNT (aryl hydrocarbon receptor nuclear transporter). Under hypoxic conditions, HIF-1α becomes stabilized and both HIF-1α and ARNT are translocated into the nucleus and codimerized, binding to the HIF-1 consensus sequence and transactivating hypoxia-inducible genes. Other than hypoxia, cobalt and nickel, which can substitute for iron in the ferroprotein, induce the stabilization of HIF-1α and the activation of HIF-1. We found previously that, although zinc, another example of a metal substitute for iron, stabilized HIF-1α, it suppressed the formation of HIF-1 by blocking the nuclear translocation of ARNT. Here, we identify a new spliced variant of human HIF-1α that is induced by zinc. The isoform lacks the 12th exon, which produced a frame-shift and gave a shorter form of HIF-1α (557 amino acids), designated HIF-1αZ (HIF-1α induced by Zn). This moiety was found to inhibit HIF-1 activity and reduce mRNA expressions of the hypoxia-inducible genes. It blocked the nuclear translocation of ARNT but not that of endogenous HIF-1α, and was associated with ARNT in the cytosol. These results suggest that HIF-1αZ functions as a dominant-negative isoform of HIF-1 by sequestering ARNT in the cytosol. In addition, the generation of HIF-1αZ seems to be responsible for the inhibitory effects of the zinc ion on HIF-1-mediated hypoxic responses, because the expressed HIF-1αZ behaved in the same manner as zinc in terms of inhibited HIF-1 activity and ARNT translocation.
Interactions between mouse ZP2 glycoprotein and proacrosin; a mechanism for secondary binding of sperm to the zona pellucida during fertilization
Authors: Howes Elizabeth , Pascall John C. , Engel Wolfgang , Jones Roy
Published: April 2021

The mouse zona pellucida glycoprotein, mZP2, is thought to be the secondary receptor on eggs for retention of acrosome-reacted sperm during fertilization. Here, we present evidence that one of its complementary binding proteins on sperm is proacrosin/acrosin. mZP2 binds to proacrosin null sperm considerably less effectively than to wild-type sperm. Binding is mediated by a strong ionic interaction between polysulphate groups on mZP2 and basic residues on an internal proacrosin peptide. The stereochemistry of both sulphate groups and basic amino acids determines the specificity of binding. Structurally relevant sulphated polymers and suramin, a polysulphonated anticancer drug, compete with mZP2 for complementary binding sites on proacrosin/acrosin in solid-phase binding assays. The same competitors also displace attached sperm from the zona pellucida of eggs in an in vitro fertilization system. This combination of genetic, biochemical and functional data supports the hypothesis that mZP2-proacrosin interactions are important for retention of acrosome-reacted sperm on the egg surface during fertilization. Safe mimetics of suramin have potential as non-steroidal antifertility agents.
Wild-type, mitochondrial and ER-restricted Bcl-2 inhibit DNA damage-induced apoptosis but do not affect death receptor-induced apoptosis
Authors: Rudner Justine , Lepple-Wienhues Albrecht , Budach Wilfried , Berschauer Johannes , Friedrich Björn , Wesselborg Sebastian , Schulze-Osthoff Klaus , Belka Claus
Published: April 2021

The proto-oncogene Bcl-2 is expressed in membranes of mitochondria and endoplasmic reticulum and mediates resistance against a broad range of apoptotic stimuli. Although several mechanisms of Bcl-2 action have been proposed, its role in different cellular organelles remains elusive. Here, we analyzed the function of Bcl-2 targeted specifically to certain subcellular compartments in Jurkat cells. Bcl-2 expression was restricted to the outer mitochondrial membrane by replacing its membrane anchor with the mitochondrial insertion sequence of ActA (Bcl-2/MT) or the ER-specific sequence of cytochrome b5 (Bcl-2/ER). Additionally, cells expressing wild-type Bcl-2 (Bcl-2/WT) or a transmembrane domain-lacking mutant (Bcl-2/ΔTM) were employed. Apoptosis induced by ionizing radiation or by the death receptors for CD95L or TRAIL was analyzed by determination of the mitochondrial membrane potential (ΔΨm) and activation of different caspases.

Bcl-2/WT and Bcl-2/MT strongly inhibited radiation-induced apoptosis and caspase activation, whereas Bcl-2/ΔTM had completely lost its anti-apoptotic effect. Interestingly, Bcl-2/ER conferred protection against radiation-induced mitochondrial damage and apoptosis similarly to Bcl-2/MT. The finding that ER-targeted Bcl-2 interfered with mitochondrial ΔΨm breakdown and caspase-9 activation indicates the presence of a crosstalk between both organelles in radiation-induced apoptosis. By contrast, Bcl-2 in either subcellular position did not influence CD95- or TRAIL-mediated apoptosis.
Evidence for a molecular complex consisting of Fyb/SLAP, SLP-76, Nck, VASP and WASP that links the actin cytoskeleton to Fcγ receptor signalling during phagocytosis
Authors: Coppolino Marc G. , Krause Matthias , Hagendorff Petra , Monner David A. , Trimble William , Grinstein Sergio , Wehland Jürgen , Sechi Antonio S.
Published: April 2021

Phagocytosis by macrophages and neutrophils involves the spatial and temporal reorganisation of the actin-based cytoskeleton at sites of particle ingestion. Local polymerisation of actin filaments supports the protrusion of pseudopodia that eventually engulf the particle. Here we have investigated in detail the cytoskeletal events initiated upon engagement of Fc receptors in macrophages. Ena/vasodilator-stimulated phosphoprotein (VASP) proteins were recruited to phagosomes forming around opsonised particles in both primary and immortalised macrophages. Not only did the localisation of Ena/VASP proteins coincide, spatially and temporally, with the phagocytosis-induced reorganisation of actin filaments, but their recruitment to the phagocytic cup was required for the remodelling of the actin cytoskeleton, extension of pseudopodia and efficient particle internalisation. We also report that SLP-76, Vav and profilin were recruited to forming phagosomes. Upon induction of phagocytosis, a large molecular complex, consisting in part of Ena/VASP proteins, the Fyn-binding/SLP-76-associated protein (Fyb/SLAP), Src-homology-2 (SH2)-domain-containing leukocyte protein of 76 kDa (SLP-76), Nck, and the Wiskott-Aldrich syndrome protein (WASP), was formed. Our findings suggest that activation of Fcγ receptors triggers two signalling events during phagocytosis: one through Fyb/SLAP that leads to recruitment of VASP and profilin; and another through Nck that promotes the recruitment of WASP. These converge to regulate actin polymerisation, controlling the assembly of actin structures that are essential for the process of phagocytosis.
SNAP-25 with mutations in the zero layer supports normal membrane fusion kinetics
Authors: Graham Margaret E. , Washbourne Philip , Wilson Michael C. , Burgoyne Robert D.
Published: April 2021

Considerable data support the idea that intracellular membrane fusion involves a conserved machinery containing the SNARE proteins. SNAREs assembled in vitro form a stable 4-helix bundle and it has been suggested that formation of this complex provides the driving force for bilayer fusion. We have tested this possibility in assays of exocytosis in cells expressing a botulinum neurotoxin E (BoNT/E)-resistant mutant of SNAP-25 in which additional disruptive mutations have been introduced. Single or double mutations of glutamine to glutamate or to arginine in the central zero layer residues of SNAP-25 did not impair the extent, time course or Ca2+-dependency of exocytosis in PC12 cells. Using adrenal chromaffin cells, we found that exocytosis could be reconstituted in cells transfected to express BoNT/E. A double Q→E mutation did not prevent reconstitution and the kinetics of single granule release events were indistinguishable from control cells. This shows a high level of tolerance of changes in the zero layer indicating that the conservation of these residues is not due to an essential requirement in vesicle docking or fusion and suggests that formation of a fully stable SNARE complex may not be required to drive membrane fusion.
Pigment epithelium-derived factor (PEDF) in neuroblastoma: a multifunctional mediator of Schwann cell antitumor activity
Authors: Crawford Susan E. , Stellmach Veronica , Ranalli Mark , Huang Xuemei , Huang Lijun , Volpert Olga , De Vries George H. , Abramson Lisa P. , Bouck Noël
Published: April 2021

Neuroblastoma is notable for its cellular heterogeneity and unpredictable outcome. Tumors are a variable mixture of primitive malignant neuroblasts, more differentiated ganglionic cells, Schwann and endothelial cells. Although often fatal, neuroblastomas can spontaneously regress, possibly due to favorable autocrine and paracrine interactions among these cells. Here, pigment epithelium-derived factor (PEDF), a potent inhibitor of angiogenesis and inducer of neural differentiation, is shown to be produced by ganglionic cells and Schwann cells, but not by more primitive tumor cells. Although undifferentiated neuroblastoma tumor cell secretions were angiogenic primarily due to vascular endothelial growth factor, secretions of Schwann cells were anti-angiogenic due to PEDF. In addition, PEDF was the major factor responsible for Schwann cell’s ability to induce tumor cell differentiation in vitro and recombinant PEDF had the same effect in vitro and in vivo. Both the growth and the survival of Schwann cells were enhanced by PEDF. Thus PEDF may serve as a multifunctional antitumor agent in neuroblastomas, inhibiting angiogenesis while promoting the numbers of Schwann cells and differentiated tumor cells that in turn produce PEDF, suggesting that its clinical administration could stimulate a multifaceted antitumor feedback loop with the potential to limit and possibly regress tumor growth.
Nesprins: a novel family of spectrin-repeat-containing proteins that localize to the nuclear membrane in multiple tissues
Authors: Zhang Qiuping , Skepper Jeremy N. , Yang Fangtang , Davies John D. , Hegyi Laszlo , Roberts Roland G. , Weissberg Peter L. , Ellis Juliet A. , Shanahan Catherine M.
Published: April 2021

In search of vascular smooth muscle cell differentiation markers, we identified two genes encoding members of a new family of type II integral membrane proteins. Both are ubiquitously expressed, and tissue-specific alternative mRNA initiation and splicing generate at least two major isoforms of each protein, with the smaller isoforms being truncated at the N-terminus. We have named these proteins nesprin-1 and -2 for nuclear envelope spectrin repeat, as they are characterized by the presence of multiple, clustered spectrin repeats, bipartite nuclear localization sequences and a conserved C-terminal, single transmembrane domain. Transient transfection of EGFP-fusion expression constructs demonstrated their localization to the nuclear membrane with a novel C-terminal, TM-domain-containing sequence essential for perinuclear localization. Using antibodies to nesprin-1, we documented its colocalization with LAP1, emerin and lamins at the nuclear envelope, and immunogold labeling confirmed its presence at the nuclear envelope and in the nucleus where it colocalized with heterochromatin. Nesprin-1 is developmentally regulated in both smooth and skeletal muscle and is re-localized from the nuclear envelope to the nucleus and cytoplasm during C2C12 myoblast differentiation. These data and structural analogies with other proteins suggest that nesprins may function as ‘dystrophins of the nucleus’ to maintain nuclear organization and structural integrity.
Fine structure of the ventral disk apparatus and the mechanism of attachment in the flagellate Giardia muris
Authors: Holberton D. V.
Published: April 2021

The topography of the Giardia trophozoite is dominated by the large domed sucking disk of the ventral surface. Attached to the host duodenal epithelium, the rim of this disk penetrates the enteric surface coat and interdigitates with microvilli of the epithelial cells, approaching to within 20 nm of the host surface membrane. Distortion of the host brush border within the disk suggests an applied suction force. A mechanical explanation of disk action is sought in a detailed description of the fine structure of components of the ventral surface - but is found to be untenable. The disk is supported by a platform of modified 25-nm microtubules, linked to the ventral membrane by side arms and bearing heavily cross-linked vertical dense ribbons. It is argued that such is the architecture of rigidity rather than relative movement.

Around the disk a mobile cytoplasmic flange is supported by 2 lateral plates of periodic substructure. The flange has no clear mechanical role in attachment; a likely evolutionary origin from a component of the anterior axonemal axis is suggested.

The cavity of the ventral disk leads posteriorly through a portal into the ventrocaudal groove: a shallow depression that houses the ventral flagella. Observation of isolated living trophozoites suggests that attachment depends on the continuing activity of the ventral flagella, which normally beat synchronously in a sinusoidal waveform. Electron micrographs confirm that this waveform is maintained in situ on the host epithelium. Of the 4 pairs of flagella, the ultrastructure of the ventral flagella is notable for additional components in the flagellar shaft, including an intraflagellar dense rod linked to 3 axonemal doublets by fine connectives.

From a consideration of analogous macroscopic systems, a preliminary hydrodynamic analysis is advanced in which the suction force of attachment follows from the pattern of fluid flow induced by the beating ventral flagella.

The significance of the conclusion that cytoplasmic microtubules (or structures derived from them) apparently maintain cell shape in the face of an applied external force is discussed.
Observations on the origin and significance of the nuclear envelopelimited monolayers of chromatin unit threads associated with the cell nucleus
Authors: Haynes M. E. , Davies H. G.
Published: April 2021

Monolayers of chromatin structural units about 33·0 nm in width enclosed on both sides by extensions of the nuclear envelope, called sheets, and located either in the cytoplasm (c· ·n· ·c type), or within the nucleus (c·· n ··n type), are common in cultured cells of Burkitt’s lymphoma. The sheets are absent from mitotic cells except at telophase where, unlike interphase, type c· ·n··n is more numerous than c· ·n··c. The degree of nuclear asymmetry is defined in terms of the increase in enclosing membranes over that required to enclose the same area in a circular configuration. The percentage number (Ps) of cells with nucleus-associated sheets averaged over all stages in the cell cycle, increases with cell viability and with nuclear asymmetry. However, during the cycle there is a marked diminution in Ps during the S-phase of DNA synthesis when nuclear asymmetry itself does not change. Hence, it is suggested, and data on other cell types support the hypothesis, that nuclear asymmetry is a necessary but not sufficient factor in causing sheets to form. Microtubules are present within the cytoplasm and their morphological arrangement suggests a role in determining nuclear asymmetry. Treatment with a microtubule depolymerizing agent, colcemid, does not alter either the existing nuclear asymmetry or Ps but when cells are treated early in S-phase the reappearance of sheets in the G2 phase of the cell cycle is considerably delayed. The reappearance takes place when the microtubules are still depolymerized. It is suggested that synthesis of membrane in excess of what is needed to enclose a sphere results in nuclear asymmetry and associated membrane-enclosed monolayers, the resulting nuclear conformation, including the distribution of membrane between types c··n· ·n and c··n··c, depending on what is energetically favoured. No biochemical function has yet been assigned to sheets.
Evidence for erythrocyte-specific histone modification and structural changes in chromatin during goose erythrocyte maturation
Authors: Brasch K. , Adams G. H. M. , Neelin J. M.
Published: April 2021

Regenerating blood of geese suffering from phenylhydrazine anaemia was separated into ‘mature’ and ‘immature’ cell populations by centrifugation through a barrier of BSA. Socalled ‘mature cells’ consisted of mainly mature erythrocytes and ‘immature cells’ included two-thirds polychromatic and younger erythroblasts. Histone proteins, dissociated from isolated nuclei of both populations of cells by sequential extraction with citric acid and hydrochloric acid, were compared and the nuclei were examined by electron microscopy. Erythrocyte-specific histone V (f2c) was fully extracted from immature nuclei at pH 2·0, but only partially extracted at the same pH from mature nuclei. An inverse correlation was found between relative ease of extraction and alkali-labile phosphate content of purified samples of histone V. The more readily dissociated fraction of histone V from immature nuclei had a higher phosphate content than the less readily dissociated component V from immature and mature nuclei. Chromatin in mature nuclei became tightly congealed after only partial extraction of histone V at pH 2·0, but loosened visibly after subsequent full extraction of histone V at pH 1·8. In contrast, chromatin in immature nuclei never became totally congealed. Histone V may be a tissue-specific agent involved in packing of DNA within chromatin fibrils. During erythropoiesis, progressive decrease of histone V phosphate may lead to its increased binding affinity for chromatin and thus to the gradual transformation of the erythrocyte genome into a permanently repressed state.
Basal body and flagellar development during the vegetative cell cycle and the sexual cycle of Chlamydomonas reinhardii
Authors: Cavalier-Smith T.
Published: April 2021

Basal body development and flagellar regression and growth in the unicellular green alga Chlamydomonas reinhardii were studied by light and electron microscopy during the vegetative cell cycle in synchronous cultures and during the sexual life cycle.

Flagella regress by gradual shortening prior to vegetative cell division and also a few hours after cell fusion in the sexual cycle. In vegetative cells basal bodies remain attached to the plasma membrane by their transitional fibres and do not act as centrioles at the spindle poles during division. In zygotes the basal bodies and associated microtubular roots and cross-striated connexions all dissolve, and by 6·5 h after mating all traces of flagellar apparatus and associated structures have disappeared. They remain absent for 6 days throughout zygospore maturation and then are reassembled during zygospore germination, after meiosis has begun.

Basal body assembly in developing zygospores occurs close to the plasma membrane (in the absence of pre-existing basal bodies) via an intermediate stage consisting of nine single A-tubules surrounding a central ‘cartwheel’. Assembly is similar in vegetative cells (and occurs prior to cell division), except that new basal bodies are physically attached to old ones by amorphous material. In vegetative cells, amorphous disks, which may possibly be still earlier stages in basal-body development occur in the same location as 9-singlet developing basal bodies. After the 9-singlet structure is formed, B and C fibres are added and the basal body elongates to its mature length. Microtubular roots, striated connexions and flagella are then assembled. Both flagellar regression and growth are gradual and sequential, the transitional region at the base of the flagellum being formed first and broken down last. The presence of amorphous material at the tip of the axoneme of growing and regressing flagella suggests that the axoneme grows or shortens by the sequential assembly or disassembly at its tip.

In homogenized cells basal bodies remain firmly attached to each other by their striated connexions. The flagellar transitional region, and parts of the membrane and of the 4 micro tubular roots, also remain attached; so also do new developing basal bodies, if present. These structures are well preserved in homogenates and new fine-structural details can be seen.

These results are discussed, and lend no support to the idea that basal bodies have genetic continuity. It is suggested that basal body development can be best understood if a distinction is made between the information needed to specify the structure of a basal body and that needed to specify its location and orientation.
The subcellular distribution of prolyl hydroxylase determined immunohistochemically
Authors: Al-Adnani M. S. , Patricko R. S. , McGEE D.
Published: April 2021

The subcellular distribution of prolyl hydroxylase was determined in rat fibroblasts and chondrocytes immunohistochemically. After fixation in 4% paraformaldehyde fibroblasts were treated with a specific goat antiscrum to rat prolyl hydroxylase followed by rabbit anti-goat IgG conjugated with horseradish peroxidase. After further fixation in glutaraldehyde the bound intracellular peroxidase activity was identified by a standard histochemical procedure. Electron microscopy of the final product showed that the antigen was located only on the membranes of the rough endoplasmic reticulum. From ultracentrifugation analysis of fibroblast homogenates 80% of the prolyl hydroxylase activity could be located in the microsomal fraction. These observations suggest that not only collagen chain synthesis but also proline hydroxylation occurs on the membranes of the rough endoplasmic reticulum.
Rifampicin inhibition of the plastid rRNA synthesis of Marchantia Polymorpha
Authors: Loiseaux S. , Mache R. , Rozier C.
Published: April 2021

The effect of rifampicin on the synthesis of plastid rRNA in Marchantia polymorpha was studied in vivo. As bacterial rRNA and plastid rRNA have the same electrophoretic mobilities, this study was possible only after a method for inhibiting bacterial contamination was developed. It was established that 91–100% of the rRNA synthesized by cultures of bacteria from Marchantia, after a labelling period of 3 and 9 h by 32P, is inhibited by 10 μg/ml of rifampicin. The same inhibition was observed when Marchantia was labelled for 3 h in the presence of 10 μg/ml of rifampicin, showing that no plastid rRNA was synthesized under out conditions, but only bacterial RNA. However, when labelling was continued for 9 h two important peaks of rRNA (23 and 16 s) were labelled in the presence of 10 or 20 μg/ml of rifampicin. These peaks are of chlorophastic origin as confirmed by the following facts: the labelling is light-activated; plastids isolated from thalli labelled for 12 h also show these two radioactive peaks. Cytoplasmic rRNA is synthesized under certain conditions. The synthesis of plastid rRNA is inhibited by higher concentrations of rifampicin, a concentration of 250 μg/ml producing at least 75% inhibition. Marchantia, a primitive multicellular plant, differs in this respect from higher plants, which seem to be, in most cases, insensitive to rifampicin.
Kinetic aspects of synchrony in suspension cultures of acer Pseudoplatanus L.
Authors: Gould A. R. , Street Η. E.
Published: April 2021

Synchronous divisions have been induced in cell suspension cultures of sycamore (Acer pseudoplatanus L.) initiated at low density from stationary phase, nitrate-starved cells. The pattern of division and DNA replication synchrony is unusual in that it does not show progressive decay but rather a sharp transition to asynchrony. Feulgen densitometry data indicate that in terms of an S-phase synchrony index, DNA replication becomes more highly synchronized as cell density rises and interphase duration is reduced. In comparison with asynchronous cultures the duration of mitosis appears to be reduced during synchronous growth. The persistence of division and S-phase synchrony and the apparent shortening of interphase and mitosis in these sycamore cultures suggests the operation of intercellular entrainment. The departure from synchrony during the interphase which precedes initiation of asynchronous division remains unexplained.
X-ray microanalysis of the mid-gut epithelium of the fruitfly Drosophila Melanogaster
Authors: Tapp R. L.
Published: April 2021

The cells in the mid-gut epithelium of Drosophila melanogasler which accumulate copper were examined by X-ray microanalysis. Both wavelength dispersive and energy dispersive analyses were used. The copper was located in granules 0.3−1.0μm in diameter which were bounded by membranes and similar, morphologically, to secondary lysosomes. The copper was associated with high concentrations of sulphur but no other elements with an atomic number greater than 9 (the lower limit of the analysis equipment) were present in appreciable concentrations.
Effects of discrete nuclear u.v.-microbeam irradiation on herpes virus and sv4o infection
Authors: Deák I. I. , Defendi V.
Published: April 2021

The requirement for a nucleolus in the expression of structural genes of nuclear viruses was examined by means of experiments in which the nucleolus or other parts of the nucleus were inactivated by a microbeam of ultraviolet light. These experiments showed that the expression of such genes is not dependent on nucleolar function. This conclusion is discussed in the light of previous experiments in which similar inactivation of the nucleolus was shown to prevent the expression of cellular structural genes.
Proteins of the newt oocyte nucleus: Analysis of the nonhistone proteins from lampbrush chromosomes, nucleoli and nuclear sap
Authors: Maundrell K.
Published: April 2021

An electrophoretic analysis has been carried out on the total protein of lampbrush chromosomes, nucleoli and nuclear sap, obtained from newt oocyte nuclei. In each case, distinctive and heterogeneous banding patterns are observed. The absence of detectable quantities of histones in oocyte chromatin is noted. In the case of the lampbrush chrómosome preparation, it is concluded that all protein species are derived from the ribonucleoprotein matrix of the lateral loops.
An ultrastructural study of the spermatozoid of the fern, Marsilea vestita
Authors: Myles Diana Gold , Bell P. R.
Published: April 2021

The ultrastructure of the mature spermatozoid of Marsilea vestita was studied after its release from the microspore and prior to its penetration of the egg. The spermatozoid is a pearshaped cell with a complex spiral structure coiled around the edge in the narrow anterior end. This coil is composed of a large mitochondrion, elongated nucleus with highly condensed chromatin, a ribbon of microtubules, and a dense band of material (flagellated band) into which the flagella are inserted. There are over a hundred flagella protruding from each spermatozoid along the length of the coil. At the anterior tip of the coil is a short multilayered structure. It is not known what maintains the helical shape of the coil. The microtubular ribbon could be involved, but it is also possible that either the flagellated band, the condensed chromatin, or both, are sufficiently rigid to retain their shapes unaided.

When the spermatozoid is first released from the microspore it includes a cytoplasmic vesicle in the posterior region containing plastids, mitochondria, and other organelles. This vesicle is shed, taking the nuclear envelope with it, before the spermatozoid reaches the egg.
Cytoplasmic streaming in Chara: A cell model activated by atp and inhibited by cytochalasin b
Authors: Williamson R. E.
Published: April 2021

After vacuolar perfusion of Chara internode cells, the cytoplasm remaining in situ can be reactivated by ATP to give full rates of streaming. Observations during both perfusion and reactivation indicated that the generation of the motive force was associated with fibres consisting of bundles of microfilaments. In the absence of ATP, the remaining endoplasmic organelles were immobilized along such fibres. When ATP was introduced, organelles moved along the fibres at speeds up to 50μms−1, but were progressively released from contact to leave the fibres in a conspicuously clean state. Inorganic pyrophosphate freed the organelles from the fibres without supporting movements. Motility required millimolar Mg2+ levels, free Ca2+ at 10−7 M or less and was inhibited by high levels of Cl− and by pH’s on either side of 7·0. The reactivated movements were rapidly and completely inhibited by 25 μ ml−1 cytochalasin B. The results are interpreted in terms of actin filaments in the stationary cortex interacting with a myosin-like protein which is able to link to endoplasmic organelles. Movement results from an active shear type of mechanism.
Ciliary granule plaques: Membrane-intercalated particle aggregates associated with Ca2+-binding sites in Paramecium
Authors: Plattner H.
Published: April 2021

In Paramecium nine rectangular aggregates of membrane-intercalated particles surround the freeze-cleaved membrane of the ciliary base. These ‘ciliary granule plaques’ occur independently of the ‘ciliary necklace’ which is observed in a more basal position on some cilia. In each individual plaque the ∼ 10 nm large granules are arranged in a square grid pattern: the granules of one plaque invariably form 3 vertical rows (number of horizontally arranged particles per row: Nh = 3) and 3–6 horizontal rows (mean value for Nv = 4 ·8); the centre-to-centre spacing of granules is dh = 22 nm in the horizontal and dv = 24 in the vertical direction. The ciliary granule plaques coincide with electron-dense deposits which can be produced in a position adjacent to the inner side of the basal ciliary shaft membrane by 3 procedures: (a) when cells are fixed in glutaraldehyde after incubation with Ca2+ concentrations (up to 25 mM) which are higher than normally present in the medium (about 0·5 HIM) but which do not immobilize the animals prior to fixation in glutaraldehyde; (b) when Ca2+ (up to 75 mM) was added to glutaraldehyde; (c) when cells were incubated with Ca2+’ as in (a) and then fixed in OsO4 solutions to which oxalate ions were added in concentrations equivalent to Ca2+. Comparable deposits were observed also with Ca2+ concentrations normally present in the medium, but they occurred much more rarely. The occurrence of electron-dense deposits was not enhanced by addition of Na+, Mg2+ or La3+ instead of Ca2+. In cross-sectioned ciliary bases the electron-dense Ca2+-dependent deposits face the 9 doublet microtubules. Occasionally the substructure of Ca2+-dependent deposits is resolved in median sections; their periodicity in the vertical direction (dv = 25nm; Nv = 5 ·2) corresponds to the periodicity of membrane-intercalated particles of freeze-cleaved plaques. Other parameters determined for Ca2+-dependent electron-dense deposits coincide also with those determined for freeze-cleaved ciliary granule plaques (values in parentheses are for cells with Ca2+ added before fixation with glutaraldehyde and ultrathin sectioning): The height of plaques (or deposits) is 84(115) nm; the distance from their lower end to the ciliary base is 138 (121) nm. Plaques (or deposits) are 45 (44) nm broad and the distance separating adjacent plaques (deposits) is 48 (50) nm. Plaques (deposits) are arranged with a horizontal periodicity of mean 93–102 (92) nm. It is concluded that the ciliary granule plaques are identical to or associated with Ca2+-binding sites involved in ciliary activity or other functions.
The effects of ionic lanthanum and hypertonic physiological salines on the nervous systems of larval and adult stick insects
Authors: Leslie R. A.
Published: April 2021

The effects of the electron-opaque tracer ionic lanthanum in various concentrations and of hyperosmotic physiological salines on the nervous system of the stick insect, Carausius morosus, have been studied.

Examination of the experimentally treated tissues revealed that the diffusion barrier to the exogenous tracer was maintained in all cases in the adult central and peripheral nervous systems, but not in the hatchling. When hatchling nervous tissues were incubated in 50 mM ionic lanthanum in hyperosmotic physiological saline, the tracer readily infiltrated all the extracellular spaces between axons and glia of all components of the nervous system examined. No difference was noted in this regard between fed and unfed hatchlings. Further, in all cases examined of adults and hatchlings, lanthanum readily surrounded those neurosecretory axons which are found in the neural lamella, or extracellular nerve sheath, of the insect.

The possible meanings of these variations in hatchling and adult nervous systems and in the accessibility of different elements of the nervous system to exogenous ionic lanthanum are discussed.
Fine structure of an organelle associated with the nucleus and cytoplasmic microtubules in the cellular slime mould Polysphondylium violaceum
Authors: Roos U. P.
Published: April 2021

Polysphondylium violaceum was grown in association with Escherichia coli. Vegetative amoebae and pseudoplasmodia were fixed under different conditions and processed for electron microscopy.

An electron-opaque body (nucleus-associated body, NAB) lies in the cytoplasm near the tapered end of interphase nuclei. The NAB consists of a disk-shaped, multilayered core, approximately 200 nm in diameter and 150 nm thick, embedded in a granular matrix from which electron-opaque nodules protrude. The nodules are termination points of microtubules radiating from the NAB into the cytoplasm or running along the nucleus. On the average there are 16 nodules per NAB. One or two microtubules terminate in each nodule. Spindle pole bodies, arising by duplication of the NAB at the beginning of mitosis, are unstructured foci for spindle microtubules in mitotic cells.

It is suggested that cytoplasmic microtubules do not determine cell shape, but they probably cause the tapering deformation of the nucleus. They may, furthermore, represent a storage form of subunits for utilization during the formation of the mitotic spindle. The nodules of the NAB are potential nucleation sites of cytoplasmic microtubules during interphase. Spindle pole bodies presumably acquire a microtubule-organizing capability by integration of the decondensed nodules.
Observations with cytochemistry and ultracryotomy on the fine structure of the expanding walls in actively elongating plant cells
Authors: Roland J. C. , Vian Brigitte , Reis Daniéle
Published: April 2021

Ultracryotomy with negative staining and cytochemistry (periodic acid – thiocarbohydrazide - silver proteinate test for polysaccharides, in conjunction with mild extractions) were used to study the architecture of the cell wall and its modifications during expansion. Those techniques were applied to the study in situ of the walls of actively elongating parenchyma of mung bean (Phaseolus aureus), and pea (Pisum sativum) root and of collenchyma of celery (Apium graveolens) petioles.

These complementary techniques provide information on the 3-dimensional disposition and fine structure of the subunits of the wall. In all the examples examined, the bulk of growing primary wall appears well-ordered and no progressive evolution from a transverse texture near the plasmalemma to a scattered texture near the middle lamella was observed.

It seems unlikely that the development of the wall structure in relation to growth could be explained mechanically by a passive shift of the fibrillar elements in response to cellular stress. There is no evidence for an inert change in fibrillar orientation in the major part of the wall. If such occurs, the process is limited to the outermost and senescent part of the wall. Thus, the texture observed does not agree with the classical multinet growth hypothesis but rather with the idea of an ordered structure of the primary wall. With the latter, the components should be able to respond in different ways to specific growth regulators and other environmental signals and thus exert a more positive control over the processes of oriented cell growth.