bluuebunny/crossref_metadata_2025 huggingface dataset:
Ventral midline cells are required for the local control of commissural axon guidance in the mouse spinal cord
Authors: Matise Michael P. , Lustig Marc , Sakurai Takeshi , Grumet Martin , Joyner Alexandra L.
Published: April 2021

Specialized cells at the midline of the central nervous system have been implicated in controlling axon projections in both invertebrates and vertebrates. To address the requirement for ventral midline cells in providing cues to commissural axons in mice, we have analyzed Gli2 mouse mutants, which lack specifically the floor plate and immediately adjacent interneurons. We show that a Dbx1 enhancer drives tau-lacZ expression in a subpopulation of commissural axons and, using a reporter line generated from this construct, as well as DiI tracing, we find that commissural axons projected to the ventral midline in Gli2−/− embryos. Netrin1 mRNA expression was detected in Gli2−/− embryos and, although much weaker than in wild-type embryos, was found in a dorsally decreasing gradient. This result demonstrates that while the floor plate can serve as a source of long-range cues for C-axons in vitro, it is not required in vivo for the guidance of commissural axons to the ventral midline in the mouse spinal cord. After reaching the ventral midline, most commissural axons remained clustered in Gli2−/− embryos, although some were able to extend longitudinally. Interestingly, some of the longitudinally projecting axons in Gli2−/− embryos extended caudally and others rostrally at the ventral midline, in contrast to normal embryos in which virtually all commissural axons turn rostrally after crossing the midline. This finding indicates a critical role for ventral midline cells in regulating the rostral polarity choice made by commissural axons after they cross the midline. In addition, we provide evidence that interactions between commissural axons and floor plate cells are required to modulate the localization of Nr-CAM and TAG-1 proteins on axons at the midline. Finally, we show that the floor plate is not required for the early trajectory of motoneurons or axons of the posterior commissure, whose projections are directed away from the ventral midline in both WT and Gli2−/− embryos, although they are less well organized in Gli2−/−mutants.
FGF signalling plays similar roles in development and regeneration of the skeleton in the brittle star Amphiura filiformis
Authors: Czarkwiani Anna , Dylus David V. , Carballo Luisana , Oliveri Paola
Published: April 2021

Regeneration as an adult developmental process is in many aspects similar to embryonic development. Although many studies point out similarities and differences, no large-scale, direct and functional comparative analyses between development and regeneration of a specific cell type or structure in one animal exist. Here, we use the brittle star Amphiura filiformis to characterise the role of the FGF signalling pathway during skeletal development in embryos and arm regeneration. In both processes, we find ligands expressed in ectodermal cells that flank underlying skeletal mesenchymal cells, which express the receptors. Perturbation of FGF signalling showed inhibited skeleton formation in both embryogenesis and regeneration, without affecting other key developmental processes. Differential transcriptome analysis finds mostly differentiation genes rather than transcription factors to be downregulated in both contexts. Moreover, comparative gene analysis allowed us to discover brittle star-specific differentiation genes. In conclusion, our results show that the FGF pathway is crucial for skeletogenesis in the brittle star, as in other deuterostomes, and provide evidence for the re-deployment of a developmental gene regulatory module during regeneration.
A combination of Notch signaling, preferential adhesion and endocytosis induces a slow mode of cell intercalation in the Drosophila retina
Authors: Blackie Laura , Tozluoglu Melda , Trylinski Mateusz , Walther Rhian F. , Schweisguth François , Mao Yanlan , Pichaud Franck
Published: April 2021

Movement of epithelial cells in a tissue occurs through neighbor exchange and drives tissue shape changes. It requires intercellular junction remodeling, a process typically powered by the contractile actomyosin cytoskeleton. This has been investigated mainly in homogeneous epithelia, where intercalation takes minutes. However, in some tissues, intercalation involves different cell types and can take hours. Whether slow and fast intercalation share the same mechanisms remains to be examined. To address this issue, we used the fly eye, where the cone cells exchange neighbors over ∼10 h to shape the lens. We uncovered three pathways regulating this slow mode of cell intercalation. First, we found a limited requirement for MyosinII. In this case, mathematical modeling predicts an adhesion-dominant intercalation mechanism. Genetic experiments support this prediction, revealing a role for adhesion through the Nephrin proteins Roughest and Hibris. Second, we found that cone cell intercalation is regulated by the Notch pathway. Third, we show that endocytosis is required for membrane removal and Notch activation. Taken together, our work indicates that adhesion, endocytosis and Notch can direct slow cell intercalation during tissue morphogenesis.
The trophoblast clock controls transport across placenta in mice
Authors: Demarez Cécile , De Assis Leonardo Vinicius Monteiro , Krohn Markus , Ramella Nahuel , Schwaninger Markus , Oster Henrik , Astiz Mariana
Published: April 2021

In mammals, 24-h rhythms of physiology and behavior are organized by a body-wide network of clock genes and proteins. Despite the well-known function of the adult circadian system, the roles of maternal, fetal and placental clocks during pregnancy are poorly defined. In the mature mouse placenta, the labyrinth zone (LZ) is of fetal origin and key for selective nutrient and waste exchange. Recently, clock gene expression has been detected in LZ and other fetal tissues; however, there is no evidence of a placental function controlled by the LZ clock. Here, we demonstrate that specifically the trophoblast layer of the LZ harbors an already functional clock by late gestation, able to regulate in a circadian manner the expression and activity of the xenobiotic efflux pump, ATP-binding cassette sub-family B member 1 (ABCB1), likely gating the fetal exposure to drugs from the maternal circulation to certain times of the day. As more than 300 endogenous and exogenous compounds are substrates of ABCB1, our results might have implications in choosing the maternal treatment time when aiming either maximal/minimal drug availability to the fetus/mother.
Collective nuclear behavior shapes bilateral nuclear symmetry for subsequent left-right asymmetric morphogenesis in Drosophila
Authors: Shin Dongsun , Nakamura Mitsutoshi , Morishita Yoshitaka , Eiraku Mototsugu , Yamakawa Tomoko , Sasamura Takeshi , Akiyama Masakazu , Inaki Mikiko , Matsuno Kenji
Published: April 2021

Proper organ development often requires nuclei to move to a specific position within the cell. To determine how nuclear positioning affects left-right (LR) development in the Drosophila anterior midgut (AMG), we developed a surface-modeling method to measure and describe nuclear behavior at stages 13-14, captured in three-dimensional time-lapse movies. We describe the distinctive positioning and a novel collective nuclear behavior by which nuclei align LR symmetrically along the anterior-posterior axis in the visceral muscles that overlie the midgut and are responsible for the LR-asymmetric development of this organ. Wnt4 signaling is crucial for the collective behavior and proper positioning of the nuclei, as are myosin II and the LINC complex, without which the nuclei fail to align LR symmetrically. The LR-symmetric positioning of the nuclei is important for the subsequent LR-asymmetric development of the AMG. We propose that the bilaterally symmetrical positioning of these nuclei may be mechanically coupled with subsequent LR-asymmetric morphogenesis.
Mutation of an Arabidopsis Golgi membrane protein ELMO1 reduces cell adhesion
Authors: Kohorn Bruce D. , Zorensky Frances D. H. , Dexter-Meldrum Jacob , Chabout Salem , Mouille Gregory , Kohorn Susan
Published: April 2021

Plant growth, morphogenesis and development involve cellular adhesion, a process dependent on the composition and structure of the extracellular matrix or cell wall. Pectin in the cell wall is thought to play an essential role in adhesion, and its modification and cleavage are suggested to be highly regulated so as to change adhesive properties. To increase our understanding of plant cell adhesion, a population of ethyl methanesulfonate-mutagenized Arabidopsis were screened for hypocotyl adhesion defects using the pectin binding dye Ruthenium Red that penetrates defective but not wild-type (WT) hypocotyl cell walls. Genomic sequencing was used to identify a mutant allele of ELMO1 which encodes a 20 kDa Golgi membrane protein that has no predicted enzymatic domains. ELMO1 colocalizes with several Golgi markers and elmo1−/− plants can be rescued by an ELMO1-GFP fusion. elmo1−/− exhibits reduced mannose content relative to WT but no other cell wall changes and can be rescued to WT phenotype by mutants in ESMERALDA1, which also suppresses other adhesion mutants. elmo1 describes a previously unidentified role for the ELMO1 protein in plant cell adhesion.
Automated in vivo drug screen in zebrafish identifies synapse-stabilising drugs with relevance to spinal muscular atrophy
Authors: Oprişoreanu Ana-Maria , Smith Hannah L. , Krix Sophia , Chaytow Helena , Carragher Neil O. , Gillingwater Thomas H. , Becker Catherina G. , Becker Thomas
Published: April 2021

Synapses are particularly vulnerable in many neurodegenerative diseases and often the first to degenerate, for example in the motor neuron disease spinal muscular atrophy (SMA). Compounds that can counteract synaptic destabilisation are rare. Here, we describe an automated screening paradigm in zebrafish for small-molecule compounds that stabilize the neuromuscular synapse in vivo. We make use of a mutant for the axonal C-type lectin chondrolectin (chodl), one of the main genes dysregulated in SMA. In chodl−/− mutants, neuromuscular synapses that are formed at the first synaptic site by growing axons are not fully mature, causing axons to stall, thereby impeding further axon growth beyond that synaptic site. This makes axon length a convenient read-out for synapse stability. We screened 982 small-molecule compounds in chodl chodl−/− mutants and found four that strongly rescued motor axon length. Aberrant presynaptic neuromuscular synapse morphology was also corrected. The most-effective compound, the adenosine uptake inhibitor drug dipyridamole, also rescued axon growth defects in the UBA1-dependent zebrafish model of SMA. Hence, we describe an automated screening pipeline that can detect compounds with relevance to SMA. This versatile platform can be used for drug and genetic screens, with wider relevance to synapse formation and stabilisation.
An ultrastructural and radioautographic study of the evolution of the interphase nucleus in plant meristematic cells (Allium porrum)
Authors: Lafontaine J. G. , Lord A.
Published: April 2021

Radioautography under both light and electron microscopy was exploited to investigate the structural changes of the chromatin reticulum which characterizes the interphase nucleus of a number of plants. Allium porrum meristematic plant cells were used for this purpose. In this species, the telophase chromosomes uncoil into dense strands which, during the G\ period, gradually give rise to a coarse reticulum. There then follows an extensive unravelling of portions of these strands, and high-resolution radioautography reveals that labelling with tritiated thymidine predominantly occurs over zones of the nucleus consisting of diffuse fine fibrillar material. As the S-period progresses, a chromatin reticulum reappears throughout the nuclear cavity, the tortuous strands being approximately 0-25 /μm in diameter. Most of the radioautographic grains still remain over the light nucleoplasmic areas but a number of these are now located on the outermost portion of the dense chromatin profiles. By the end of the S-period, the chromatin strands are slightly thicker (ca. 0-3 μm) and form a looser reticulum. Labelling has decreased noticeably in nuclei of that period, the radioautographic grains being grouped into clusters resting over more or less spherical regions of the chromatin reticulum. Judging from their localization at the surface of the nucleolus or close to the nuclear envelope, these structures correspond to chromocentres. The additional interesting finding that such nuclear structures appear much less compactly organized strongly suggests that chromocentres undergo important conformational modifications during duplication of their DNA.
Some properties of ribosome crystals isolated from hypothermically treated chick embryos
Authors: Dondi P. G. , Barker D. C.
Published: April 2021

Ribosome crystals observed in hypothermically treated chick embryos exist as large dense aggregates and, for this reason, could be purified from a post-nuclear supernatant by centrifugation through a high-density discontinuous sucrose gradient.

A ribosome crystal fraction isolated in this way was observed, by electron microscopy, to contain intact crystals, resembling those seen in sectioned whole cells, with only minor contamination from morphologically indistinct particles.

The ribonucleic acid (RNA) content of this fraction was found to be the same as ribosomal RNA from mature chickens. However, their resistance to mild RNase treatment, lack of transfer RNA and endogenous messenger RNA activity indicate that such ribosome crystals are not a specific type of polysome.

The intertetramer bonds (i.e. bonds between tetramers in a crystal array) are much more susceptible to mild protease treatment than to treatment with ribonuclease (RNase), indicating a dependence on protein-protein interactions for their formation.
The role of endoplasmic reticulum in the repair of amoeba nuclear envelopes damaged microsurgically
Authors: Flickinger C. J.
Published: April 2021

The nuclear envelopes of amoebae were damaged microsurgically, and the fate of the lesions was studied with the electron microscope. Amoebae were placed on the surface of an agar-coated slide. Using a glass probe, the nucleus was pushed from an amoeba, damaged with a chopping motion of the probe, and reinserted into the amoeba. Cells were prepared for electron microscopy at intervals of between 10 min and 4 days after the manipulation. Nuclear envelopes studied between 10 min and 1 h after the injury displayed extensive damage, including numerous holes in the nuclear membranes. Beginning 15 min after the manipulation, pieces of rough endoplasmic reticulum intruded into the holes in the nuclear membranes. These pieces of rough endoplasmic reticulum subsequently appeared to become connected to the nuclear membranes at the margins of the holes. By 1 day following the injury, many cells had died, but the nuclear membranes were intact in those cells that survived. The elaborate fibrous lamina or honeycomb layer characteristic of the amoeba nuclear envelope was resistant to early changes after the manipulation. Patches of disorganization of the fibrous lamina were present 5 h to 1 day after injury, but the altered parts showed evidence of progress toward a return to normal configuration by 4 days after the injury. It is proposed that the rough endoplasmic reticulum participates in the repair of injury to the nuclear membranes. The similarity of this repair process to reconstitution of the nuclear envelope in telophase of mitosis is noted, and the relationship between the nuclear envelope and the rough endoplasmic reticulum is discussed.
Ultrastructural observations of cell wall regeneration around isolated tobacco protoplasts
Authors: Burgess J. , Fleming E. N.
Published: April 2021

The process of cell wall regeneration around cultured protoplasts isolated from tobacco mesophyll has been examined by electron microscopy. The initially formed wall contains 2 components which stain with conventional heavy metal stains. The first consists of unbranched fibres, at first oriented at right angles to the plasmalemma surface. As wall growth proceeds the fibres lengthen and assume an orientation parallel to the plasmalemma. It seems probable that this component is cellulose. The second component of the wall is more amorphous and more densely stained. It is most frequently seen in situations where leaching of materials into the medium would be expected to be minimal. The endoplasmic reticulum and the plasmalemma are the only membrane systems which appear to contribute towards wall formation. No pattern of structure has been detected to explain the orientation or method of synthesis of the microfibrillar part of the wall.
Accumulation of gene products in the oocytes of the dragonfly Cordulia aenea l.: I. the nematosomes
Authors: Halkka Liisa , Halkka O.
Published: April 2021

The chromosomes of the active previtellogenic oocytes of the dragonfly Cordulia aenea L. extrude into the cytoplasm a substance, which is subsequently found there in the form of granulofibrillar masses. These nuclear extrusions evolve ultrastructurally into two components, nematosomes and dense masses. The nematosomes are later found transitorily adjacent to the mitochondria and undergo transformations that are synchronized with changes in oocyte activity. It is suggested that the nematosomes are storage structures for long-lived informational RNA and that they may be widely distributed in various types of cells containing such RNA.
Nucleocytoplasmic transport and distribution of an amino acid, in situ
Authors: Frank M. , Horowitz S. B.
Published: April 2021

Ultra-low temperature techniques (microdissection and autoradiography) were used to study the nucleocytoplasmic distribution and transport of α-aminoisobutyric acid (AIB) in an amino acid-accumulating cell.

In amphibian oocytes incubated in AIB, the nuclear concentration of this non-metabolizable amino acid exceeds the cytoplasmic concentration by 45 %, remaining constant both over time and with variation in substrate concentration. The kinetics of uptake suggest that this nucleo-cytoplasmic asymmetry arises from solubility differences between the 2 compartments, and that the nuclear envelope plays a negligible role in amino acid transport.

A solute exclusion model is offered to explain the nucleocytoplasmic asymmetry.
Cellular uptake of soluble and aggregated ferritin: Distinction between pinocytosis and phagocytosis
Authors: Petitpierre-Gabathuler M.-P. , Ryser H. J.-P.
Published: April 2021

Cellular uptake of ferritin amounting to 0·5 μg/mg cell protein or more can be measured colorimetrically on the basis of ferritin-iron content. 131I-serum albumin, soluble ferritin and aggregated ferritin used in equimolar concentrations are taken up differently by Sarcoma S180 cells in culture. The net uptakes in 2 h at 37 °C are 0·065, 4·3 and 24·7 μg I mg cell protein or 0·93, 8·0 and 45·7 μmol, respectively. Albumin uptake is not inhibited by a 26-fold molar ferritin excess but is significantly inhibited by a 43-fold excess.

The transport mechanism of the ferritins differs from that of albumin in that it is significantly inhibitable by 2 × 10−1 M monoiodoacetate. Soluble ferritin contains small aggregates which are removed by filtration through Millipore membranes of 0·05, 01 and 0·22 μm. When the 0·1μm filtrate is re-examined, uptake is no longer inhibited by iodoacetate. Since it can be inferred from other work that albumin is taken up by pinocytosis and ferritin aggregates by phagocytosis, the difference in susceptibility to inhibition is proposed as a way to distinguish pinocytosis from phagocytosis.

Ferritin may form larger visible aggregates in culture medium. The transport mechanism of this aggregated ferritin differs from that of soluble unfiltered ferritin in that it causes concomitant enhancement of albumin uptake. Albumin transported by virtue of this effect becomes partially susceptible to iodoacetate. Thus, in addition to a distinction between pinocytosis and phagocytosis, our data single out 2 forms of albumin transport and 3 forms of ferritin transport.
Localization of lectins in legume cotyledons
Authors: Clarke A. E. , Knox R. B. , Jermyn M. A.
Published: April 2021

High-resolurion techniques for the localization of lectins are described. Concanavalin A (Con A) and phytohaemagglutinin (PHA) are localized using a fluorescent method with (FITC)-labelled immunoglobulins which bind to the lectins in sections of jack and red kidney bean cotyledons. Specificity is defined by the use of specific sugar inhibitors. Both Con A and PHA are found in cytoplasmic sites.

Lectins with β-glycoside specificity are detected with red-coloured artificial carbohydrate antigens. The β-galactosyl and β-glucosyl antigens bind specifically to clusters of spherical bodies in the intercellular spaces, to cell wall sites, and to the periphery of the cytoplasm associated with the cell membrane.
Circus movement in dissociated embryonic cells of a teleost,Oryzias latipes
Authors: Fujinami Noboru , Kageyama Tetsuo
Published: April 2021

The dissociated early embryonic cells of the fresh water fish, Oryzias latipes, protrude hyaline lobopodia, which tend to rotate around the cell circumference in a propagating wave. Cells from late blastula or gastrula continuously show this ‘circus movement’, while most cells up to early blastula are rounded. The linear velocity of the lobopodium was estimated by means of time-lapse cinemicrography. The velocity increases slightly as cell diameter increases.

The effects of pH, temperature and osmotic pressure of the immersion media on the movement were also quantitatively investigated. Cells become rounded and do not form lobopodial blebs when immersed in media below pH 5. The velocity is reduced by decreasing temperature, but the movement continues even at 5 °C. Cells placed in hypertonic salt solutions become crenated and do not continuously demonstrate the circus movement.
A model of pattern formation in insect embryogenesis
Authors: Meinhardt H.
Published: April 2021

A model is proposed in which the interaction of an autocatalytic substance with a short diffusion range - the activator - and its more diffusible antagonist - the inhibitor - leads to a local high concentration of activator at the posterior pole of the egg. The inhibitor, which is then produced mainly in this activated region, diffuses into the rest of the egg, where it acts as a ‘morphogen forming a concentration gradient which supplies positional information.

This model can account quantitatively for the patterns resulting from a large number of different experiments performed during early insect development, including ligation, u.v.- irradiation and microsurgical manipulations. The formation of additional posterior structures is interpreted as the result of the appearance of a new activator peak. Omission of segments after ligation of the egg is explained as the result of accumulation of morphogen (the inhibitor) at the posterior side of the ligation and a decrease of morphogen on the anterior side. In order to account for certain quantitative features of the ligation experiments it is necessary to assume that determination in response to the morphogen gradient is a slow, stepwise process, in which the nuclei or cells first pass through determination stages characteristic for more anterior structures until they ultimately form a given structure.

The mutual interactions of activator and inhibitor are expressed as a set of partial differential equations. The individual experiments are simulated by solving these equations by use of a computer.
An ultrastructural study of the relationship between the plasma membrane and the cell wall of the coenocytic alga Hydrodictyon africanum
Authors: Bailey D. S. , Northcote D. H.
Published: April 2021

A wall/plasma membrane unit was prepared from Hydrodictyon africanum by microdissection. A replica of the inner surface of the membrane was made and by freeze-fracture of the whole cell, 2 corresponding internal fracture faces were obtained. The large coenocytes of the alga were plasmolysed and the wall separated by cutting it away. Its inner surface was directly viewed in the electron microscope after shadowing with Pt/C. Particles were found on the outer half of the internal fracture face of the membrane which were oriented in the same 2 directions as the microfibrils laid down at the inner surface of the wall. No structures were found at the inner surface of the membrane. Some evidence was obtained for a structural connexion between the innermost layers of the wall and the plasma membrane.
Mitotic spindles of Drosophila melanogaster: A phase-contrast and scanning electron-microscope study
Authors: Milsted Amy , Cohen William D. , Lampen Nina
Published: April 2021

Mitotic spindles have been isolated from the blastema stage of Drosophila melanogaster embryos using modified tubulin-polymerizing medium. ‘Clean’ spindles, relatively free of contaminating cytoplasmic material, are obtained. Under phase contrast, mitotic stages appear remarkably similar to those seen in situ, as reported in early literature. This preservation of morphological integrity, coupled with relative structural simplicity due to low chromosome number (2n = 8), makes these spindles ideal subjects for study. Use of the scanning electron microscope provides excellent visulization of their general structural organization, changes in whole spindle structure during the course of mitosis, and higher resolution viewing of surface detail than is permitted with light microscopy.
Gibberellic acid-induced germination of spores of Anemia phyllitidis; autoradiographic study of the timing and regulation of nucleic acid and protein synthesis in relation to cell morphogenesis
Authors: Raghavan V.
Published: April 2021

The timing and regulation of nucleic acid and protein synthesis during gibberellic acid-induced germination of spores of the fern, Anemia phyllitidis (L.) Swartz were investigated using metabolic inhibitors combined with cytological and autoradiographic techniques. Incubation of spores in gibberellic acid for at least 24 h was necessary for their subsequent germination in the absence of the hormone. Based on the action of cycloheximide, it appeared that continuous synthesis of proteins was essential for the hydrolysis of storage reserves, enlargement of the protoplast, division of the spore nucleus and formation of the protonemal cell associated with germination. Addition of actinomycin D or 5-fluorouracil along with gibberellic acid did not visibly affect germination, although the latter inhibited division of the spore nucleus. Both compounds also inhibited RNA synthesis in germinating spores without any noticeable effect on DNA or protein synthesis. Cordycepin was most effective in inhibiting germination when it was added during the first 24 h of gibberellic acid action. Spores visibly germinated in the absence of nuclear division when DNA synthesis was inhibited by hydroxyurea. On the other hand, incubation of spores in an osmoticum such as mannitol led to no discernible signs of germination, although division of the nucleus and formation of the protonemal cell occurred in the absence of cell enlargement. It is tentatively proposed that the dry spore contains a stable mRNA which is activated in the presence of gibberellic acid. This mRNA provides templates for the synthesis of proteins necessary for the early events of germination.
The role of nuclear Ca2+ in maintaining neuronal homeostasis and brain health
Authors: Mozolewski Pawel , Jeziorek Maciej , Schuster Christoph M. , Bading Hilmar , Frost Bess , Dobrowolski Radek
Published: April 2021

Nuclear Ca2+ has emerged as one of the most potent mediators of the dialogue between neuronal synapses and the nucleus that regulates heterochromatin states, transcription factor activity, nuclear morphology and neuronal gene expression induced by synaptic activity. Recent studies underline the importance of nuclear Ca2+ signaling in long-lasting, activity-induced adaptation and maintenance of proper brain function. Diverse forms of neuroadaptation require transient nuclear Ca2+ signaling and cyclic AMP-responsive element-binding protein (CREB1, referred to here as CREB) as its prime target, which works as a tunable switch to drive and modulate specific gene expression profiles associated with memory, pain, addiction and neuroprotection. Furthermore, a reduction of nuclear Ca2+ levels has been shown to be neurotoxic and a causal factor driving the progression of neurodegenerative disorders, as well as affecting neuronal autophagy. Because of its central role in the brain, deficits in nuclear Ca2+ signaling may underlie a continuous loss of neuroprotection in the aging brain, contributing to the pathophysiology of Alzheimer's disease. In this Review, we discuss the principles of the ‘nuclear calcium hypothesis’ in the context of human brain function and its role in controlling diverse forms of neuroadaptation and neuroprotection. Furthermore, we present the most relevant and promising perspectives for future studies.
Calcium exchangeability in subcellular fractions of pancreatic islet cells
Authors: Borowitz J. L. , Matthews E. K.
Published: April 2021

The distribution and exchangeability of calcium within subcellular compartments of rat pancreatic islet cells was determined in pulse-chase experiments following isotopic labelling with 45Ca2+. In unstimulated islets the mitochondrial fraction showed the greatest, and the insulin secretory granule fraction the least, uptake of 45Ca2+; subsequently the rate of loss from each compartment during 120 min was in the order mitochondrial > microsomal > secretory granule fraction. On exposure to D-glucose, 16·8 mM, the 45Ca labelling of all 3 subcellular compartments was increased after 5 and 30 min stimulation. In contrast, theophylline 10 mM caused a significant decrease in mitochondrial 45Ca content with little effect on other compartments. These results are discussed in relation to the importance of calcium disposition, exchangeability, and mobility for the initiation and control of insulin secretion.
The effect of chloramphenicol, ethidium bromide and cycloheximide on mortality and mitochondrial protein synthesis of adult blowflies
Authors: Ashour Badr , Tribe Michael , Whittaker Peter
Published: April 2021

The effects of cycloheximide, chloramphenicol and ethidium bromide on the blowfly Calliphora erythrocephala were studied. In the first set of experiments, toxic levels were determined by examining activity and mortality of flies after injection of various doses of each drug. In the second set of experiments, the effect of drug treatment on flight muscle mitochondrial protein synthesis was determined in relation to age by following the incorporation of radioactively labelled amino acid, [3H]leucine, into mitochondrial protein in vivo. To confirm the developmental changes in flight muscle mitochondria, mitochondrial protein content per fly was estimated from emergence to 30 days of age; the highest protein level was recorded between 6 and 10 days of age.

Maximum incorporation of labelled amino acid was found in newly emerged flies, and this age group was also the most sensitive to drug treatment. By the time flies had reached 6 –10 days of age, amino acid incorporation had declined to about two-thirds of the rate obtained with newly emerged flies. With 6-10-day-old flies, however, the highest value for flight muscle mitochondrial protein per fly was recorded, and these flies also displayed the greatest resistance to drug treatment of any age group investigated. For example, inhibition of protein synthesis following injection of 300 μg/fly of chloramphenicol was only about 15 % below the untreated control in 6-10-day-old flies, whereas in all other age groups investigated, inhibition ranged between 30 and 50 % of the controls.

At 15 –20 days of age, protein synthesis decreased to a third of the newly emerged flies’ rate and continued to decrease further in the 30 –35-day-old group, where it was less than one sixth of the youngest age group. The effect of drug treatment on these older flies was also less than that observed with newly emerged flies, especially after chloramphenicol and ethidium bromide injections. The effect of cycloheximide however, was much the same in all age groups, with inhibition of protein synthesis being 80—90 % of controls.

Surprisingly, cycloheximide (1 –10 μg/fly) had little initial effect on mortality of young flies, despite almost complete blockage in the synthesis of mitochondrial proteins at these concentrations. 95 % mortality occurred only when doses of 20 μg/fly were given. In contrast, high doses of chloramphenicol (400 μg/fly) and ethidium bromide (15 μg/fly) caused almost total mortality a few hours after injection, although such doses never induced more than about 50 % inhibition of mitochondrial protein synthesis.

Each drug therefore has a different site of inhibition and induces different mortality effects. Possible explanations for these differences in mortality are discussed.
Ultrastructure of polytene chromosomes of Drosophila isolated by microdissection
Authors: Mott Margaret R. , Burnett E. J. , Hill R. J.
Published: April 2021

Drosophila polytene chromosomes prepared by a new micromanipulative procedure, which avoids acid squashing, have been examined at the ultrastructural level in the electron microscope. Puffs at 2B, 68C, 74EF, 75B and 85EF, have been examined in some detail, along with the chromocentre and various interbands. The ultrastructure of these chromosomes, which have never been exposed to acid protein denaturants, compares favourably with that of classical acid-fixed specimens. Ribonucleoprotein particles in puffs are seen to be organized in linear arrays and evidence is adduced for looped transcription units. Particles with characteristic sizes and morphologies are observed near the chromocentre, in puffs and in interbands. In interbands RNP particles and ‘superbead’-like chromatin particles may be distinguished. Drosophila polytene chromosomes isolated by micro-manipulation should prove useful for the localization of native chromosomal proteins at an ultrastructural level.
Measurement and characteristics of fusion of isolated membrane fractions from maize root tips
Authors: Baydoun Elias A-H. , Northcote D. H.
Published: April 2021

Maize root tips were incubated in vivo with radioactive glucose, choline or diazotized sulphanilic acid. Membrane fractions were prepared from radioactive and non-radioactive roots. The transfer of radioactivity between mixed membrane fractions has enabled a quantitative system to be developed to study in vitro membrane fusion between Golgi apparatus and plasma membrane-rich fractions. Membrane fusion was found to be dependent on time, temperature, Mn2+ and Ca2+. Mn2+ was as effective as Ca2+, other divalent cations had a moderate or no effect. The effect of various substances, including inhibitors of microtubular and microfilament assembly and blocking reagents for sulphydryl group on membrane fusion has been investigated. The process appears to be dependent on the membrane proteins or glycoproteins; trypsinization of mixed membranes prior to the addition of Ca2+ inhibited significantly the fusion process. SDS-polyacrylamide gel electrophoresis of trypsin-treated membranes revealed the selective loss of one particular polypeptide which could play a role in membrane fusion.
Hierarchy of fibrillar organization levels in the polytene interphase chromosomes of Chironomus
Authors: Sass Heinz
Published: April 2021

The present study shows a hierarchy of fibrillar organization levels in polytene somatic interphase chromosomes. The chromosomes of several Chironomus species have been examined by electron microscopy in situ, i.e., in the physiological environment of intact salivary glands, and in vitro as spread preparations. In situ, the basic structural element of bands, interbands and constitutive heterochromatin is a DNP fibre (chromatid) which varies in thickness from 11 to 25 nm. The fibre displays a varying degree of coiling in bands and interbands. It runs more or less straight through the interbands but forms aggregates (chromomeres), which have a densely packed fibrillar texture, in the band regions. On the whole there is always an abrupt transition between the fibre and the chromomeres which it connects. Constitutive heterochromatin is also made up of a fibre of the same thickness as in bands and interbands. However, in the heterochromatin the fibre generally measures 20–25 nm. In telomeric heterochromatin the fibre is extremely densely arranged, whereas the centric heterochromatin (chromocentre) of C. melanotus is made up of very loosely bundled fibres. Neither type of heterochromatin exhibits any visible subdivision into chromomeres and interchromomeres.

In spread preparations of native isolated polytene chromosomes of different Chironomus tissues seen in vitro the bands and interbands are also composed of a DNP fibre which varies in thickness from 11 to 25 nm. Fibres in spread preparations are at most 20–25 nm thick. The spreading of band regions enables one to recognize more or less tangled, sometimes transcribed 0 · 8–8 · 4 μm loops, which are regarded as unfolded chromomeres. The shape of the 20–25-nm fibre in band (loop) and interband regions is irregular and clearly reflects orderly packing of globular particles (superbeads) embedded in a diffuse matrix. The latter can be removed by spreading the fibres in low salt medium or distilled water, which also makes individual superbeads visible. Arranged in a row, 4 superbeads account for 0·1 μm. Superbeads are structures consisting of subunits which correspond in size (n 0± 10 nm) to nucleosomes. Tentatively, there are 7–12 nucleosomes in one superbead. A row of nucleosomes constitutes the 11–13-nm fibre. This is the basic fibre of polytene chromosomes. The 20–25-nm fibre appears to be a higher order of the basic fibre. Digestion of the 20–25-nm fibre by trypsin and DNase reveals that the fibre is unineme as far as its DNA is concerned. The fact that polytene interphase chromosomes analysed in situ and in vitro both show a 20-25-nm fibre, leads to the conclusion that this fibre represents the highest level of fibrillar organization of the DNP fibre. Presumably the distinct levels of organization revealed by the 20–25-nm fibre in vitro also exist in the chromosomes in situ, but are invisible because the fibres prepared at physiological ionic strength are densely packed.
The role of negative charge in spontaneous aggregation of transformed and untransformed cell lines
Authors: Wright Thomas C. , Smith Barton , Ware Ben R. , Karnovsky Morris J.
Published: April 2021

No correlation between net negative surface charge as determined by electrophoretic light-scattering techniques and the rates of spontaneous aggregation of 3T3MIT and SVPy 3T3MIT cells has been found. Neuraminidase treatment of both 3T3MIT cells and SVPy 3T3MIT cells causes a significant decrease in electrophoretic mobility but only the 3T3MIT cells show an increase in spontaneous aggregation. An increase in spontaneous aggregation of 3T3MIT cells is seen after growth in 200 mM urea for 18 h but no change in net surface charge occurs.

The distribution of anionic sites on the membranes of cells was determined using the ultrastructural marker polycationized ferritin. The distribution of polycationized ferritinbinding sites was essentially identical for both cell lines under all conditions when they were labelled at 4 °C. When the cells were labelled with polycationized ferritin at 37 °C it was found that cells which have a high net rate of spontaneous aggregation also show rearrangement of anionic sites on their surface membrane. Clustering and rearrangement of anionic sites at 37 °C correlate with high rates of spontaneous aggregation.
Immunofluorescent histological studies of the role of fibronectin in the expression of the associative preferences of embryonic tissues
Authors: Armstrong Peter B. , Armstrong Margaret T.
Published: April 2021

The identity of the chemical factors controlling the spreading behaviour of sheets of cells was examined in organ culture. When aggregates of two dissimilar tissues are apposed in organ culture, one tissue spreads reproducibly over the surface of the second. The present study employed indirect immunofluorescent localization techniques to evaluate the hypothesis that the spreading behaviour of chick embryonic heart tissue in culture is dominated by the presence or absence of the cell-surface and extracellular matrix protein fibronectin in the surface layers of the aggregates. Specifically, the hypothesis proposes that aggregates that display surface fibronectin earlier after culturing and/or in higher quantities segregate internally to aggregates that are slower to develop a surface layer of fibronectin or in which this layer contains reduced amounts of fibronectin. The hypothesis has been supported for 3 categories of behaviour of chick embryo heart tissue: (1) myocyte aggregates spread over myocyte aggregates containing a 20% admixture of heart fibroblasts, which in turn spread over heart fibroblast aggregates; (2) S-day embryonic ventricle-tissue fragments maintained in culture for 0μ·5 days spread over ventricle fragments cultured for 2·5 days; and (3) 2-day embryonic ventricle spreads over 5-day ventricle. In all these situations, the aggregate type that segregates to an internal position displays more fibronectin at its surface than aggregate types that spread to occupy an external position. Evidence is presented that the fibronectin in heart tissue aggregates is elaborated by heart fibroblasts.
The proportion of ribosomes engaged in rat pancreatic polysomal structures as influenced by pilocarpine injection
Authors: Poort C. , Kramer M. F.
Published: April 2021

Rats intravenously injected with pilocarpine at a concentration of 4 mg per 100 g body weight were killed after various time intervals and the relative numbers of ribosomes per cell active in protein synthesis were determined. It was found that until 1 h after pilocarpine injection there is a significant decrease in the number of ribosomes engaged in protein synthesis, whereas 2–3 h after pilocarpine injection, the number of active ribosomes equals that of control animals. After a short incubation in vitro, the same relative number of active ribosomes is always found, independent of the value determined in the pancreas directly after the death of the animal.
A search for synaptonemal complexes in Ustilago maydis
Authors: Fletcher H. L.
Published: April 2021

Germinating spores, germ-tubes (promycelia), gall tissue and spores developing in the gall were examined. No synaptonemal complexes (SCs) were found in any of these cell types. There are 3 possible explanations for this: (1) Ustilago maydis does not have SCs. This is the case in Schizosaccharomyces pombe (Olson, Eden, Egel-Mitani & Egel, 1978) and in many strains of Saccharomyces cerevisiae (e.g. see Byers & Goetch, 1975). (2) The SC might occur in spores either in the gall or before germination when the spore wall was too solid to allow examination of the contents by the methods used. (3) The SC was present, but for a very short time, and most of the cells examined were in any case not undergoing meiosis, so the SCs were not seen. (Large numbers of spores were examined, but few spores germinate and undergo normal meiotic division; also a minority of the cells in the gall are forming spores at any one time.) The gall was found to contain uninucleate single cells (i.e. it is a yeast, as it is in artificial culture medium) and virtually all these cells were haploid, 1 in 3000 recovered cells was diploid. It appears that the haploids fuse to form a heterokaryotic or diploid cell immediately before spore formation. A heterokaryotic phase is presumed to exist to establish and maintain the infection.
Intracellular distribution of nadph-cytochrome c reductase in rat hepatocytes studied by direct ferritin-immunoelectron microscopy
Authors: Aoi Kazuo , Fujii-Kuriyama Yoshiaki , Tashiro Yutaka
Published: April 2021

NADPH-cytochrome c reductase was purified from rat liver microsomes and the monospecific antibodies to the reductase were prepared from the antiserum by affinity chromatography using immunoadsorbent gel. Ferritin was coupled to the specific antibodies and the approximately equimolar conjugates were isolated by gel filtration. By direct ferritin-immuno-electron microscopy, using these conjugates, it was revealed that the ferritin particles are localized exclusively on the microsomal vesicles and the outer nuclear envelope. In contrast, binding of ferritin particles to Golgi membranes, outer mitochondrial membranes and plasma membranes was slight and at control level. On each microsomal vesicle, the ferritin particles were distributed heterogeneously, sometimes forming clusters. An assay of the binding of equimolar conjugates with microsomes showed that microsomes bind ∼ 1 mol of antibody per mol of reductase. From these data the maximum number of ferritin particles that can bind with microsomes was calculated. This number is in agreement with the average number of ferritin particles bound per microsome, as determined experimentally by observing a number of crosssectional profiles of microsomal vesicles previously incubated with the conjugates at saturation level. This showed that the distribution of the reductase could be analysed semi-quantitatively by the present ferritin-immunoelectron-microscopical analyses. It was also shown that smooth microsomes can bind more conjugates than rough microsomes. The average number of ferritin particles on each microsomal vesicle increased in proportion to the increase in the amount of reductase in the microsomes after treatment with phenobarbital. Finally, the non-random distribution of ferritin particles on microsomal vesicles was confirmed by statistical analysis of electron micrographs of a number of the labelled microsomes.
Reconstruction of complexes of histone and superhelical nuclear dna
Authors: Levin Jeremy M. , Cook Peter R.
Published: April 2021

When HeLa cells are lysed in solutions containing a non-ionic detergent and 2 M-NaCl, structures are released that retain many of the morphological features of nuclei. These nucleoids contain all the nuclear RNA and DNA but few of the proteins characteristic of chromatin. Their DNA is supercoiled and so intact. Using a simple and rapid procedure we have reconstructed nucleohistone complexes from nucleoids and the ‘core’ histones without breaking the DNA. We have probed the integrity and structure of the reconstructed complexes using a non-destructive fluorometric approach, which provides a general method for detecting agents that bind to DNA and alter its supercoiling. The βuperhelical status of the DNA in the reconstructed complexes is indistinguishable from that found in control nucleoids containing core histones. Experiments with micrococcal nuclease confirm that the DNA in the reconstructed complexes is organized into nucleosome-like structures. These, however, are spaced 145 base-pairs apart and not 200 base-pairs apart as is found in native chromatin.