bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of protein kinase Cη triggers cortical granule exocytosis inXenopusoocytes
Authors: Gundersen Cameron B. , Kohan Sirus A. , Chen Qian , Iagnemma Joseph , Umbach Joy A.
Published: April 2021

Previous work has shown that phorbol esters or diacylglycerol trigger cortical granule exocytosis in Xenopus oocytes. We sought to identify the isoform(s) of protein kinase C (PKC) that mediate(s) this regulated secretory event. Because this process is initiated by lipid activators of PKC but is independent of calcium ions, we focused on the family of novel(calcium-independent) PKCs. Pharmacological investigations using Gö6976 and Gö6983 tended to exclude PKCδ, ϵ and μ as secretory triggers. Subcellular fractionation and immunoblot data revealed that these oocytes expressed all five members of the novel PKC family, but it was only PKCη that colocalized with cortical granules. Finally, expression of wild type or constitutively active forms of PKCδ and η strongly supported the conclusion that it is PKCη that initiates cortical granule exocytosis in these cells. These observations represent an important step in identifying the mechanism of secretory triggering in this system.
Musashi: a translational regulator of cell fate
Authors: Okano Hideyuki , Imai Takao , Okabe Masataka
Published: April 2021

Transcription is thought to have a major role in the regulation of cell fate; the importance of translational regulation in this process has been less certain. Recent findings demonstrate that translational regulation contributes to cell-fate specification. The evolutionarily conserved, neural RNA-binding protein Musashi, for example, controls neural cell fate. The protein functions in maintenance of the stem-cell state, differentiation, and tumorigenesis by repressing translation of particular mRNAs. In mammals it might play an important role in activating Notch signalling by repressing translation of the Notch inhibitor m-Numb.
Protein phosphatase 4 is required for centrosome maturation in mitosis and sperm meiosis inC. elegans
Authors: Sumiyoshi Eisuke , Sugimoto Asako , Yamamoto Masayuki
Published: April 2021

The centrosome consists of two centrioles surrounded by the pericentriolar material (PCM). In late G2 phase, centrosomes enlarge by recruiting extra PCM,and concomitantly its microtubule nucleation activity increases dramatically. The regulatory mechanisms of this dynamic change of centrosomes are not well understood. Protein phosphatase 4 (PP4) is known to localize to mitotic centrosomes in mammals and Drosophila. An involvement of PP4 in the mitotic spindle assembly has been implicated in Drosophila, but in vivo functions of PP4 in other organisms are largely unknown. Here we characterize two Caenorhabditis elegans PP4 genes, named pph-4.1 and pph-4.2. Inhibition of the function of each gene by RNA-mediated interference (RNAi) revealed that PPH-4.1 was essential for embryogenesis but PPH-4.2 was not. More specifically, PPH-4.1 was required for the formation of spindles in mitosis and sperm meiosis. However, this phosphatase was apparently dispensable for female meiotic divisions, which do not depend on centrosomes. In the cell depleted of pph-4.1 activity,localization of γ-tubulin and a Polo-like kinase homologue to the centrosome was severely disturbed. Immunofluorescence staining revealed that PPH-4.1 was present at centrosomes from prophase to telophase, but not during interphase. These results indicate that PPH-4.1 is a centrosomal protein involved in the recruitment of PCM components to the centrosome, and is essential for the activation of microtubule nucleation potential of the centrosome. Furthermore, chiasmata between homologous chromosomes were often absent in oocytes that lacked pph-4.1 activity. Thus, besides promoting spindle formation, PPH-4.1 appears to play a role in either the establishment or the maintenance of chiasmata during meiotic prophase I.
Co-regulation of cell adhesion by nanoscale RGD organization and mechanical stimulus
Authors: Koo Lily Y. , Irvine Darrell J. , Mayes Anne M. , Lauffenburger Douglas A. , Griffith Linda G.
Published: April 2021

Integrin-mediated cell adhesion is central to cell survival,differentiation and motility. Many cell responses induced by integrins require both receptor occupancy and receptor aggregation, and appear to be regulated by both biochemical and biophysical means. Multidomain extracellular matrix molecules may serve to foster integrin aggregation by presenting local clusters of adhesion ligands, a hypothesis supported by studies with synthetic substrates showing that cell adhesion and migration are enhanced when adhesion ligands are presented in nanoscale clusters. Here, we used a novel synthetic polymer system to present the adhesion ligand GRGDSPK in nanoscale clusters with 1.7, 3.6 or 5.4 peptides per cluster against a non-adhesive background,where the peptide is mobile on a 2 nm polyethylene oxide tether. Average ligand density ranged from 190 to 5270 RGD/μm2. We used these substrates to study the effects of ligand density and clustering on adhesion of wild-type NR6 fibroblasts, which expressα vβ3 andα 5β1, integrins known to bind to linear RGD peptides. The strength of cell-substratum adhesion was quantified using a centrifugal detachment assay to assess the relative number of cells remaining adherent after a 10 minute application of defined distraction force. An unusual relationship between cell detachment and distraction force at relatively low values of applied force was found on substrates presenting the clustered ligand. Although a monotonic decrease in the number of cells remaining attached would be expected with increasing force on all substrates,we instead observed a peak (adhesion reinforcement) in this profile for certain ligand conditions. On substrates presenting clustered ligands, the fraction of cells remaining attached increased as the distraction force was increased to between 70 and 150 pN/cell, then decreased for higher forces. This phenomenon was only observed on substrates presenting higher ligand cluster sizes (n=3.6 or n=5.4) and was more pronounced at higher ligand densities. Adhesion reinforcement was not observed on fibronectin-coated surfaces. These results support previous studies showing that biophysical cues such as ligand spatial arrangement and extracellular matrix rigidity are central to the governance of cell responses to the external environment.
Extracellular signal regulated kinase (ERK)/mitogen activated protein kinase (MAPK)-independent functions of Raf kinases
Authors: Hindley Alison , Kolch Walter
Published: April 2021

Raf comprises a family of three kinases, A-Raf, B-Raf and Raf-1, which are best known as key regulators of the MEK—MAPK/ERK cascade. This module is often perceived as a linear pathway in which ERK is the effector. However,recent advances have unveiled a role for Raf outside this established signalling unit. Current evidence, including gene-knockout studies in mice,suggests that there are ERK-independent functions of Raf kinases. Regulation of apoptosis is one area in which Raf may function independently of ERK,although its substrates remain to be identified. Other studies have suggested that Raf has kinase-independent functions and may act as a scaffold protein.
The importin-β P446L dominant-negative mutant protein loses RanGTP binding ability and blocks the formation of intact nuclear envelope
Authors: Timinszky Gyula , Tirián László , Nagy Ferenc T. , Tóth Gábor , Perczel András , Kiss-László Zsuzsanna , Boros Imre , Clarke Paul R. , Szabad János
Published: April 2021

Three of the four independently induced KetelDdominantnegative female sterile mutations that identify the Drosophila importin-β gene, originated from a C4114→ T transition and the concurrent replacement of Pro446 by Leu (P446L). CD spectroscopy of representative peptides with Pro or Leu in the crucial position revealed that upon the Pro→Leu exchange the P446L mutant protein loses flexibility and attains most likely an open conformation. The P446L mutation abolishes RanGTP binding of the P446L mutant form of importin-β protein and results in increased RanGDP binding ability. Notably, the P446L mutant importin-β does not exert its dominant-negative effect on nuclear protein import and has no effect on mitotic spindle-related functions and chromosome segregation. However, it interferes with nuclear envelope formation during mitosis-to-interphase transition, revealing a novel function of importin-β.
Robust G1 checkpoint arrest in budding yeast: dependence on DNA damage signaling and repair
Authors: Gerald Jonathan N. Fitz , Benjamin Jacqueline M. , Kron Stephen J.
Published: April 2021

Although most eukaryotes can arrest in G1 after ionizing radiation, the existence or significance of a G1 checkpoint in S. cerevisiae has been challenged. Previous studies of G1 response to chemical mutagens, X-ray or UV irradiation indicate that the delay before replication is transient and may reflect a strong intra-S-phase checkpoint. We examined the yeast response to double-stranded breaks in G1 using γ irradiation. G1 irradiation induces repair foci on chromosome spreads and a Rad53 band shift characteristic of activation, which suggest an active DNA damage response. Consistent with a G1 arrest, bud emergence, spindle pole duplication and DNA replication are each delayed in a dose-dependent manner. Sensitivity to mating pheromone is prolonged to over 18 hours when G1 cells are lethally γ or UV irradiated. Strikingly, G1 delay is the predominant response to continuousγ irradiation at a dose that confers no loss of viability but delays cell division. Like the G2/M checkpoint, G1 delay is completely dependent on both RAD9 and RAD24 epistasis groups but independent of POLϵ. Lethally irradiated rad9 mutants rapidly exit G1 but perform a slow S phase, whereas rad17 and rad24 mutants are completely arrest deficient. Distinct from γ irradiation, G1 arrest after UV is RAD14 dependent, suggesting that DNA damage processing is required for checkpoint activation. Therefore, as in the yeast G2/M checkpoint response, free DNA ends and/or single-stranded DNA are necessary and sufficient to induce a bona fide G1 checkpoint arrest.
Stem cells of the oesophageal epithelium
Authors: Seery John P.
Published: April 2021

Cancers arising in the oesophageal epithelium are among the most common fatal tumors in the world. Despite this, comparatively little is known about the cell biology and organization of this tissue. Recently, in vitro and in vivo techniques developed over the past 30 years for the study of the epidermis have been applied to the study of the oesophageal epithelium. This approach, combined with data from previous histochemical studies, has lead to the identification and isolation of putative oesophageal epithelial stem cells. Oesophageal epithelial stem cells demonstrate several unusual properties, and their identification may facilitate studies on oesophageal carcinogenesis.
Increased responsiveness of hypoxic endothelial cells to FGF2 is mediated by HIF-1α-dependent regulation of enzymes involved in synthesis of heparan sulfate FGF2-binding sites
Authors: Li Jian , Shworak Nicholas W. , Simons Michael
Published: April 2021

Binding of basic fibroblast growth factor (FGF2) to its high affinity receptors requires the presence of specific heparan sulfate (HS) moieties on the cell surface that act as coreceptors. To determine the contribution of cell-surface HS to modulation of FGF2-dependent cell growth, we studied the changes in the cell mass and FGF2 binding of endothelial cell HS under normoxic and hypoxic conditions in vitro. Both large vein and cardiac microvascular endothelial cells cultured under hypoxic conditions demonstrated an increase in the ratio of cell-surface HS to chondroitin sulfate (CS), as well as an increase in the number of low affinity (HS-associated) binding sites for FGF2 with no change in the apparent Kd. This increase in the number of HS-FGF2 binding sites, in the absence of a significant change in FGF receptor expression, resulted in enhanced responsiveness of hypoxic,compared with normoxic, endothelial cells to FGF2 stimulation.

Gene expression studies demonstrated increased expression of the key regulatory enzyme responsible for HS chain synthesis, 1,4 GlcNAc transferase(GlcNAcT-I), as well as increased expression of 2-O sulfotransferase (HS2ST),the enzyme responsible for sulfation of IdoA, a crucial part of the HS-FGF2 binding site. Transduction of cells with an adenovirus encoding a HIF-1αexpression construct resulted in a similar increase in GlcNAcT-I and HS2ST expression. We conclude that hypoxia increases endothelial cell responsiveness to FGF2 by promoting preferential synthesis of HS rather than CS chains and increasing the number of FGF2-binding sites on HS chains. Both of these events are mediated by a HIF-1α-dependent increase in expression of the enzymes GlnNAcT-I and HS2ST. This shift in cell-surface HS composition results in enhanced cell sensitivity to FGF2-induced growth stimulation.
The catalytic domain of endogenous urokinase-type plasminogen activator is required for the mitogenic activity of platelet-derived and basic fibroblast growth factors in human vascular smooth muscle cells
Authors: Padró Teresa , Mesters Rolf M. , Dankbar Berno , Hintelmann Heike , Bieker Ralf , Kiehl Michael , Berdel Wolfgang E. , Kienast Joachim
Published: April 2021

Emerging data suggest that urokinase-type plasminogen activator (UPA),beyond its role in pericellular proteolysis, may also act as a mitogen. We investigated the function of endogenous UPA in mediating the mitogenic effects of platelet-derived growth factor (PDGF) and basic fibroblast growth factor(bFGF) on human vascular smooth muscle cells (SMC). Growth-arrested SMC constitutively expressed UPA, but UPA expression and secretion increased several times upon stimulation with either PDGF or bFGF. Inhibition of endogenous UPA with a polyclonal antibody significantly reduced DNA synthesis and proliferation of PDGF or bFGF stimulated SMC, this effect already being evident when the cells entered S-phase. The proliferative activity of endogenous UPA was dependent on a functional catalytic domain as demonstrated by inhibition experiments with a specific monoclonal antibody (394OA) and p-aminobenzamidine, respectively. In contrast, neither plasmin generation nor binding of UPA to its receptor (CD87) were required for UPA-mediated mitogenic effects. The results demonstrate that endogenous UPA is not only overexpressed in SMC upon stimulation with PDGF/bFGF, but also mediates the mitogenic activity of the growth factors in a catalytic-domain-dependent manner. Specific inhibition of this UPA domain may represent an attractive target for pharmacological interventions in atherogenesis and restenosis after angioplasty.
Ca2+-binding proteins of cilia and infraciliary lattice ofParamecium tetraurelia: their phosphorylation by purified endogenous Ca2+-dependent protein kinases
Authors: Kim Kwanghee , Son Min , Peterson Joan B. , Nelson David L.
Published: April 2021

We purified two small, acidic calcium-binding proteins(ParameciumCa2+-binding proteins, PCBP-25α and PCBP-25β) from Paramecium tetraurelia by Ca2+-dependent chromatography on phenyl-Sepharose and by anion-exchange chromatography. The proteins were immunologically distinct. Monoclonal antibodies against PCBP-25β did not react with PCBP-25α, and antibodies against centrin from Chlamydomonas reacted with PCBP-25α but not with PCBP-25β. Like the centrins described previously, both PCBPs were associated with the infraciliary lattice (ICL), a fibrillar cytoskeletal element in Paramecium. Both were also present in isolated cilia, from which they could be released (with dynein) by a high-salt wash, and both PCBPs cosedimented with dynein in a sucrose gradient. PCBP-25β was especially prominent in cilia and in the deciliation supernatant, a soluble fraction released during the process of deciliation. The results of immunoreactivity and localization experiments suggest that PCBP-25α is a Paramecium centrin and that PCBP-25β is a distinct Ca2+-binding protein that confers Ca2+ sensitivity on some component of the cilium, ciliary basal body or ICL.

We characterized these proteins and Paramecium calmodulin as substrates for two Ca2+-dependent protein kinases purified from Paramecium. PCBP-25α and calmodulin were in vitro substrates for one of the two Ca2+-dependent protein kinases (CaPK-2), but only PCBP-25α was phosphorylated by CaPK-1. These results raise the possibility that the biological activities of PCBP-25α and calmodulin are regulated by phosphorylation.
Meiosis in Coprinus: IV. morphology and behaviour of spindle pole bodies
Authors: Raju N. B. , Lu B. C.
Published: April 2021

Meiotic synchrony in the genus Coprinus has permitted the sequential study of spindle pole body (SPB) behaviour through the meiotic process. The SPBs are monoglobular in the young basidia immediately after the last premeiotic mitosis. From 10 to 15 h before karyogamy until pachytene, spindle pole bodies are not found. They become conspicuous in diplotene and persist until the completion of meiosis. During diplotene, metaphase, anaphase and telophase stages the spindle pole bodies are monoglobular but at late diplotene they duplicate and become diglobular with an isthmus connecting the 2 globular elements. The spindle pole bodies remain in a diglobular state until diakinesis when the isthmus breaks separating the 2 daughter spindle pole bodies. The diglobular SPBs in late diplotene and prophase II are believed to represent the duplicated form of the monoglobular state. The spindle pole bodies in Coprinus contain no centrioles. In thin sections the SPBs appear to be fibrillar amorphous structures with a dense inner core surrounded by a less-dense outer zone.
The early development of muscle spindles in the rat
Authors: Milburn Alice
Published: April 2021

The morphogenesis of muscle spindles in rat lower hind-limb muscles has been investigated using the electron microscope. The earliest detectable spindles are seen in the ig-5-day foetus and consist of a single myotube bearing simple nerve terminals of the large primary afferent axon from nearby unmyelinated intramuscular nerve trunks. The capsule forms by an extension of the perineural epithelium of the supplying nerve fasciculus, and is confined initially to the innervated zone. Myonuclei accumulate in this region, so that the first intrafusal muscle fibre to develop is a nuclear-bag fibre. Myoblasts, present within the capsule of the spindle throughout its development, fuse to form a smaller less-differentiated myotube by the 20-day foetal stage. This new myotube matures by close association with the initial fibre, and by birth (21–22 days gestation) has formed the smaller, intermediate bag fibre, that has been identified histochemically and ultra-structurally in the adult. The nuclear-chain fibres develop in the same way; myoblasts fuse to form satellite myotubes that mature in pseudopodial apposition to one of the other fibres within its basement membrane. This apposition consists of extensions of sarcoplasm from the developing myotube into the supporting fibre. By the 4-day postnatal stage the full adult complement of 4 intrafusal muscle fibres is present, although ultra-structural variations, seen in the adult, are not differentiated. The fusimotor innervation begins to arrive at birth, but is not mature until the 12th postnatal day, when the myofibrillar ultra-structural differentiation, including the loss of the M-line in the large-diameter bag fibre, is complete. The periaxial space appears at the same time. It is suggested that the sequential development of the intrafusal fibres is a reflexion of the decreasing morphogenetic effect of the afferent innervation, whereas the role of the fusimotor innervation is in ultra-structural, myofibrillar differentiation.
Functional development of the reticular system in an insect muscle with synchronously differentiating cells
Authors: Tyrer N. M.
Published: April 2021

In contrast to many developing muscles which contain a spectrum of cells at different stages of differentiation, cell development in the locust abdominal intersegmental muscle tends to be synchronous, so that at any stage of development the cells are at the same stage of differentiation. This makes it feasible to relate the properties of the developing tissue as a whole to changes occurring within the cells.

Muscle contraction in the abdominal dorsal muscles of locust embryos and young hoppers has been related to ultra-structural changes within the muscle. Early in development both contraction and relaxation rates are very slow and the muscle shortens very little. The maximum registered tension is achieved relatively early while the rates of contraction and relaxation remain slow. Contraction and relaxation become more rapid as the embryo develops.

These changes can be related to the development of the reticular system, various components of which mature at different rates. When the muscle is first able to develop tension the myofibres contain scattered clusters of myofilaments while the transverse tubule system (T-system), the cisternae and the sarcoplasmic reticulum (SR) are not well developed. The myofibres reach their final size and the filaments are fully formed while the T-system is still irregular and the SR is sparse. The T-system and the cisternae become well developed before the SR. During the time that the relaxation time decreases the SR becomes increasingly prominent.
The substructure of ciliary microtubules
Authors: Warner F. D. , Satir P.
Published: April 2021

Gill cilia from the freshwater mussel Elliptio complanatus were examined after negative staining with phosphotungstic acid. The doublet and central pair microtubules typically collapse on the grid supporting film so that the protofilaments making up the wall of each tubule spread into a visible array. Thirteen protofilaments can be seen to form both the central pair microtubules and subfibre A of each doublet. Sub fibre B of the doublet consists of 10 proto-filaments.

As viewed in both normal and thiourea-treated cross-sectioned cilia, subfibre A appears as a complete microtubule with a circular profile and 26 nm diameter. Subfibre B is an incomplete, elliptically profiled tubule that shares, as a common wall or partition, 4 or 5 protofilaments of the A subfibre. The organization of the 3·5-nm thick protofilaments within the microtubules and their relationship to the various doublet appendages is discussed.
Microfilaments and cytoplasmic streaming: Inhibition of streaming with cytochalasin
Authors: Bradley M. O.
Published: April 2021

Cytochalasin B reversibly inhibits cytoplasmic streaming in both Nitella and Avena cells. Colchicine, on the other hand, has no effect on streaming in either plant; nor does colchicine prevent the recovery of streaming after cytochalasin is withdrawn. The inhibition of protein synthesis by cycloheximide has no effect on either streaming itself or on the recovery of streaming after cytochalasin withdrawal. All this suggests that microfilaments may provide one component of the structure that generates the streaming force and that microtubules play little, if any, role in the process.

Ultrastructural studies of Nitella demonstrate that microfilaments are localized at the boundary of the streaming endoplasm and the stationary ectoplasm. Microfilaments are organized in discrete bundles, with possible cross-bridges between individual filaments in each bundle. These bundles are closely associated with the extensive endoplasmic reticulum.

Cytochalasin B does not cause ultrastructural changes in Nitella microfilaments as it does in some animal-cell filaments. Since the molecular mechanism of cytochalasin’s action is unknown, there may be no necessary correlation between functional inhibition by the drug and altered microfilament morphology.

A model is advanced which proposes that streaming is generated by an interaction between microfilaments and the endoplasmic reticulum.
An ultrastructural and radioautographic study of early oogenesis in the toad Xenopus Laevis
Authors: Coggins Lesley Watson
Published: April 2021

Early oogenesis in the toad Xenopus laevis has been investigated at the ultrastructural level, with particular reference to the formation of extrachromosomal DNA. Thymidine incorporation was localized by electron microscope radioautography. In oogonia, the nucleus is irregular in outline and may contain several nucleoli. Oocytes, from premeiotic interphase to late pachytene, are found in cell nests which are estimated to consist of about 16 cells each. Adjacent oocytes within a nest are connected by intercellular bridges and develop synchronously. Each premeiotic interphase-leptotene oocyte has a round nucleus which contains one or two centrally located, spherical nucleoli. Electron-microscope radioautography showed that all nuclei in a cell nest incorporate thymidine synchronously during premeiotic S-phase. In zygotene oocytes, axial cores and synaptonemal complexes are observed in the nucleus and abut against the inner nuclear membrane in the region nearest the centre of the cell nest. The nucleolus is still more- or-less round in outline, but is asymmetrically positioned in the nucleus. It lies near the nuclear envelope on the side of the nucleus furthest away from the attachment of the chromosome ends, that is, nearest the outside of the cell nest. Each nucleolus is surrounded by a fibrillar ‘halo’ of nucleolus-associated chromatin into which a low level of thymidine incorporation occurs during zygotene. This is thought to represent the start of the major period of amplification of the ribosomal DNA. Pachytene is characterized by the presence of synaptonemal complexes in the nucleus. The nucleolus becomes very irregular in outline. The fibrillar area around it, which represents the extrachromosomal DNA, increases in size and thymidine is incorporated over the whole of this region. In late pachytene, many small fibrogranular bodies, the multiple nucleoli, are formed in it. The members of a cell nest become separated from one another at this time and begin to develop asynchronously. In diplotene, synaptonemal complexes are no longer observed in the nucleus. The most prominent structures in the nucleus are now the multiple nucleoli, which increase greatly in number in early diplotene. A large increase in cytoplasmic volume occurs and the oocyte grows in size.
An ultrastructural study of the differentiation of the spermatozoid of Equisetum
Authors: Duckett J. G.
Published: April 2021

The ultrastructure of spermatogenesis in Equisetum is described with particular reference to the origin and development of the multilayered structure (MLS) and nuclear metamorphosis. Simultaneously with the formation of centrioles, by the fragmentation of the blepharoplast, in young spermatids, the MLS appears in their vicinity. This comprises 4 layers recalling the Vierergruppe of bryophyte spermatids. The outer layer, or microtubular band, consists of juxtaposed microtubules. The three inner lamellar strata, which lie along the anterior edge of the microtubular band, are composed of parallel plates oriented at 35-45° to the axes of the microtubules. Keels are present on the microtubules where these overlie the lamellar layers. A mitochondrion lies subjacent to the lamellar layers and on the outer surface of the anterior edge of the microtubular band is a crest of osmiophilic material. The position of the osmiophilic crest suggests that it may have a role in microtubule synthesis. However, its persistence in the mature gametes after microtubular elongation has ceased, and its banded substructure, reminiscent of flagellar roots, perhaps indicate that its function is mainly mechanical in holding the microtubular band together. Approximately oval in shape and overlain by less than 50 short microtubules initially, the lamellar strata and subjacent mitochondrion rapidly increase in length. Eventually they form a strip 15-20 μm in length overlain by over 300 microtubules. This extensive microtubular band in Equisetum is more likely related to the final shape of the nucleus in the mature gamete than to the presence of numerous flagella. The entire MLS now becomes associated with the nucleus. The microtubular band is closely adpressed to the nuclear envelope and acts as a cytoskeletal framework along which the nucleus undergoes elongation and coiling. Initially the lamellar strip and mitochondrion run along the nuclear envelope with one of their edges touching it and the other projecting into the cytoplasm. However, continuous elongation of the microtubules throughout nuclear metamorphosis results in the gradual separation of the strip and mitochondrion beyond the anterior tip of the nucleus. Simultaneously, the posterior parts of the nucleus become ensheathed by rearward extension of the microtubular band. The centrioles arrange themselves in a single layer on the outer surface of the microtubular band and during the early stages of nuclear metamorphosis give rise to flagella from their distal ends, concomitantly undergoing differentiation into basal bodies. Intense Golgi activity during early and mid-spermatid stages is thought to be related to the accumulation of mucopolysaccharides between the cell wall and cell membrane. In the mid-spermatids rough endoplasmic reticulum is closely associated with the plastids which later accumulate starch, a characteristic feature of spermatogenesis in archegoniate plants.
Autoradiographic evidence for the rapid disintegration of one chloroplast in the zygote of the green alga Ulva Mutabilis
Authors: Bråten T.
Published: April 2021

This paper gives evidence suggesting that one of the two chloroplasts contributed to the zygote of Ulva mutabilis is rapidly destroyed. Chloroplasts in an advanced stage of disintegration can be seen only 4 min after copulation. Radioactive labelling of gametes prior to copulation shows that the disintegrating chloroplast is supplied through the + (plus) gamete, while the chloroplast from the — (minus) gamete is retained in the zygote. These observations are in agreement with genetic observations from the same alga.
Effect of anaerobiosis on respiratory rate, cytochrome oxidase activity and mitochondrial structures in coleoptiles of rice (oryza sativa l.)
Authors: Öpik Helgi
Published: April 2021

An attempt has been made to correlate respiration rate, cytochrome oxidase activity and mitochondrial structure in coleoptiles of rice, Oryza sativa L., germinated under aerobic and anaerobic conditions. The rice coleoptiles emerge from the grain and elongate considerably even under complete anaerobiosis, which totally suppresses root and leaf growth. Cell number, dry weight and nitrogen content per coleoptile are all lowered, although some cell division and translocation of reserves into the coleoptile does take place under anaerobiosis. Compared with coleoptiles from air-grown seedlings, anaerobically grown coleoptiles have a much lower capacity for respiratory oxygen uptake and their cytochrome oxidase activity is depressed even more. Mitochondria, however, are still abundant in 4-day-old anaerobic coleoptiles, with a crista density only slightly lower than in cells of aerobically grown coleoptiles. Since, in the embryonic coleoptile of the ungerminated grain, mitochondria show very little internal structure, a considerable amount of elaboration of mitochondrial structure must occur in the rice coleoptile under anaerobiosis, contrasting with the situation in yeast, where mitochondria of normal structure are formed only in aerobic conditions. Since a high crista density develops in rice coleoptile mitochondria with a very much depressed cytochrome oxidase activity, there is no obligate correlation between crista density and cytochrome oxidase activity in this tissue.
The incorporation of DL-[3-14c]cysteine during spermatogenesis inCeratopteris thalictroides
Authors: Schedlbauer M. D. , Cave Celia F. , Bell P. R.
Published: April 2021

Radioactive cysteine fed for 6 h to young gametophytes of Ceratopteris actively producing antheridia was incorporated readily into the early stages of spermatogenesis, but hardly at all into differentiating spermatocytes. Spermatocytes contained only little radioactivity even when the gametophytes were grown for a further 18 h before fixation, thus ensuring that the radioactive label was available at or near the time of their formation. There was some evidence that in very young spermatocytes in the latter material the incorporation was more marked at the periphery of the blepharoplast than elsewhere.

The results are interpreted as indicating that the differentiation of the spermatocyte involves little if any net protein synthesis. The proteins of the new structures peculiar to the gamete may thus have been synthesized in earlier stages of spermatogenesis, the last phase of visual differentiation being a consequence of their assembly and organization.
Timing of the first cleavage division of the mouse and the duration of its component stages: A study of living and fixed eggs
Authors: Kaufman M. H.
Published: April 2021

Fertilized mouse eggs were examined between 27 and 34 h after the superovulating injection of human chorionic gonadotrophin (HCG). Out of 1334 eggs examined, 432 were already at the 2-cell stage; the remaining 902 at the i-cell stage were examined in detail. All chromosome preparations of the first cleavage mitosis were classified into groups corresponding with the stages of prometaphase (early and late), metaphase (early or ‘prechromatid’, ‘chromatid’ and ‘late chromatid’) and anaphase.

An indirect estimate was made of the duration of the first cleavage mitosis and of its component stages from the incidence of stages observed at different time intervals after the HCG injection. Fertilized eggs were also observed at 37 °C by time-lapse cine-photography and the interval between the disappearance of the pronuclei and the beginning of telophase of the first cleavage division was determined. The progress of eggs fertilized in vitro was also observed under normal culture conditions. A close correlation was observed between the indirect method of assessing the mitotic time and the direct values obtained from the studies on time-lapse and in vitro culture. The effect of temperature on the mitotic time was also examined by the time-lapse method.
Sensitivity to diphtheria toxin as a species-specific marker in hybrid cells
Authors: Dendy P. R. , Harris H.
Published: April 2021

Diphtheria toxin may be used as a species-specific marker in the study of interspecific hybrid cells. Sensitivity to the toxin is dominant in heterokaryons made by fusing sensitive and insensitive cells.
Temperature-sensitive rna polymerase of a mammalian cell mutant
Authors: Naha P. M.
Published: April 2021

In our attempts to characterize a temperature-sensitive mutant (ts-2) of monkey kidney cell line BSC-1, we have provided evidence to show that the mutant was defective in the synthesis or processing of rRNA (45, 28 and 18 s) molecules at 39·5 °C and there was indication that the nucleolar RNA polymerase (I) or some factor associated with its functional activity was thermolabile at the restricted temperature of 39·5 °C. The enzyme in the wild type cell line was, however, stable at this temperature. Though conclusive evidence to implicate RNA polymerase (I) was not obtained in these experiments, temperature-sensitivity tests with crude enzyme extracts of the RNA polymerase in ts-2 showed that it contained a temperature-sensitive factor which was either degraded rapidly or failed to be eluted out of the DEAE-Sephadex column. This temperature-sensitive factor was not affected by α-amanitin.
Cryo-ultramicrotomy of islets of langerhans: some observations on the fine structure of mammalian islets in frozen sections
Authors: Howell S. L. , Tyhurst Margaret
Published: April 2021

A procedure is described for the preparation of ultrathin frozen sections of glutaraldehyde-fixed or unfixed islets of Langerhans by cryo-ultramicrotomy. Freezing of the tissue was accomplished by direct immersion of isolated islets in liquid nitrogen. Sectioning was performed at a specimen temperature of–80 °C and a knife temperature of–40 °C, the ribbon of sections being collected on a trough containing 60 % dimethyl sulphoxide. Staining was accomplished with 4 % silicotungstic acid and sections were protected from drying artifacts by rinsing with 0·5 % polyethylene glycol.

Even in tissue not subjected to prior glutaraldehyde fixation, most of the structural features of A and B cells were well preserved in frozen sections, which were obtained in a number and quality which should render them suitable for ultrastructural, cytochemical or radio-autographic studies.
Quantitative measurement of the course of bean callus differentiation
Authors: Haddon Lindsay E. , Northcote D. H.
Published: April 2021

Two strains of callus have been isolated from bean hypocotyl and grown on a defined maintenance medium supplemented with 2 mg/1. 2:4-dichlorophenoxyaceric acid (2:40) and 2% sucrose. Root initiation was observed in one strain and formation of nodules containing xylem and phloem in both strains after transfer to an induction medium supplemented with 1 mg/1. naphthyleneacetic acid, 0·2 mg/1. kinetin and 3% sucrose, after 3 transfers to maintenance medium.

The number of nodules per gramme increased 10-fold between 6 and 12 days after transfer, and thereafter remained constant. Phenylalanine ammonia lyase (PAL) activity rose to a maximum value when the rate of nodule formation was greatest, and decreased after the maximum nodule concentration was reached. The final constant value for PAL activity was above that of callus grown on maintenance medium, β →3 glucan synthetase activity rose to a maximum 15 days after transfer, and then fell gradually to a level above that measured in callus on maintenance medium.

Callus was transferred from maintenance medium after 3, 4, 5 and 6 transfers. The concentration of nodules after 21 days on induction medium decreased as the callus was kept in culture. No further differentiation could be induced after 6 transfers. The fall in nodule formation was paralleled by a decrease in PAL and β →3 glucan synthetase activities measured 21 days after transfer.
Meiosis in Coprinus vii. the prekaryogamy S-phase and the postkaryogamy dna replication in C. lagopus
Authors: Lu B. C. , Jeng D. Y.
Published: April 2021

The kinetics of incorporation of 32P into DNA have unequivocally shown that the premeiotic S-phase in Coprinus lagopus occurs before the onset of karyogamy. It takes 8 h under the control conditions (25 °C with a 16 h light-8 h dark regime) but only 6 h under the arrest-release conditions. An important discovery in this study is that the initiation of premeiotic DNA replication is subject to an arrest by restrictive conditions (35 °C under a continuous light regime) whereas that of the mitotic replication is not. Once initiated, meiotic DNA replication can continue even under the restrictive conditions. Incorporation of “P into DNA at pachytene is quite extensive. These replications are considered to be repair replications.
Cell surface lipids and adhesion: II. the turnover of lipid components of the plasmalemma in relation to cell adhesion
Authors: Curtis A. S. G. , Shaw F. M. , Spires V. M. C.
Published: April 2021

The preceding paper showed that those conditions that ought to stimulate reacylation of lysolipids in cells can increase cell adhesion. Similarly we found that conditions that would be expected to lead to the accumulation of lysolipids in the cell surface diminish cell adhesion. This paper reports on the answers to the following questions.

Is reacylation of lysolipids in the cells stimulated by an external supply of CoA, ATP and a fatty acid ? Does this reacylation lead to the incorporation of exogenous fatty acid in the plasmalemma ? What range of fatty acids can be incorporated into the plasmalemma and into what compounds? Does the plasmalemma contain the enzyme systems to effect this turnover, namely phospholipase A,, a CoA-ligase and an appropriate acyl transferase(s) ? Do lysolipids accumulate in the plasmalemma under conditions which diminish cell adhesion ?

We find that saturated fatty acids in the range C14–C18 and some unsaturated fatty acids are incorporated into the plasmalemmae of these neural retina cells. About 20 % of the plasma-lemmal content of fatty acids can be turned over in 30’. Incorporation is mainly into phosphatidyl choline, serine and ethanolamine in both R1 and R2 positions. The plasmalemmae contain the enzymes to effect the turnover. Isolated plasmalemmae are active in this turnover. Incubation of the plasmalemmae with phospholipase A2 leads to an accumulation of lysolipids. Very low levels of phospholipase stimulate turnover, possibly because endogenous phospholipase activity is the rate-limiting step in the system. These findings are discussed in relation to the possible mechanisms by which lipids might affect adhesion.
Appearances of microtubules after various fixative procedures, and comparison with the appearances of tobacco mosaic virus
Authors: Forer A. , Blecher S. R.
Published: April 2021

Microtubules in crane-fly spermatids appeared altered when the glutaraldehyde-fixed cells were not postfixed with osmium tetroxide. The cytoplasmic microtubules were altered more than the doublet microtubules. Addition of osmium tetroxide after dehydration did not produce appearances identical with those of microtubules postfixed directly after glutaraldehyde, and thus at least some alterations occurred during dehydration, possibly due to extraction of microtubule-associated lipid. The omission of osmium tetroxide postfixation did not cause drastic alterations in the appearances of either tobacco mosaic virus (TMV), or polymerized tobacco mosaic virus protein (without RNA), suggesting that microtubule stability is different from TMV stability (with respect to the embedment procedure). The electron-dense stain associated with embedded–sectioned TMV is predominantly outside the TMV protein, as demonstrated by the known distribution of TMV protein compared with the dimensions of sectioned TMV and negatively stained TMV. The same might hold true for microtubules, as evidenced by the dimensions of negatively stained, isolated brain microtubules compared with those of embedded and sectioned brain microtubules.
Light-microscopic observations of individual microtubules reconstituted from brain tubulin
Authors: Kuriyama Ryoko , Miki-Noumura Taiko
Published: April 2021

The course of polymerization of individual brain microtubules could be observed with a light microscope employing dark-field illumination. Statistical analysis of the increase in microtubule length during the polymerization was in accordance with the time course of viscosity change of the tubulin solution. After a plateau level in viscosity was attained, there was no significant change in histograms showing length distribution. These observations were confirmed with fixed and stained microtubules, using a phase-contrast microscope.

Observations with dark-field illumination revealed that reconstituted microtubules depolymerized and disappeared immediately upon exposure to buffer containing CaCl2 or sulphydryl reagents such as p-chloromercuriphenyl sulphonic acid (PCMPS) and p -chloromercuribenzoic acid (PCMB). They were also cold-labile. The growth of heterogeneous microtubules which were assembled by mixing purified tubulin dimers with ciliary outer fibres could also be followed with these optical systems.
Intranuclear electrophoresis of the chromatin of living cells
Authors: Skaer R. J. , Whytock Susan , Emmines J. P.
Published: April 2021

Intranuclear electrophoresis of living cells under appropriate conditions causes the chromatin and nucleoli to move rapidly into the anodal side of the nucleus. In pig kidney cells, chromatin lengths attached to the nuclear envelope are oriented by the current and freed from surrounding non-oriented chromatin. Individual chromatin strands isolated in this way are often long and have not been subjected to the trauma of isolation from the nucleus. This has allowed us to demonstrate oriented lines of up to 8 chromomeres in a strand, linked by fine single fibres. These chromomeres of chromatin have the same linear dimensions as the bands and interbands of polytene chromosomes. A very wide range of morphology of chromatin is revealed — from lines of nucleosomes in open array, to strands uniformly 25 nm thick. Doublet strands and multiple strands — often embedded in darkly staining material — are also seen. All morphological types may be seen in the same nucleus. Many of the oriented threads appear to be transcriptionally active. The variable morphology of these sites and their relation to peripheral heterochromatin is discussed. Histone nucleosomes are present in these apparently transcriptionally active regions. The method is useful for investigating the relationships between chromatin and the nuclear envelope. Approximately 1500 attachment sites per nucleus are found in these cells. Some nucleoli are attached to the nuclear envelope.
Amino acid metabolism of myeloma cells in culture
Authors: Roberts R. S. , Hsu H. W. , Lin K. D. , Yang T. J.
Published: April 2021

The growth of myeloma cells in Leibovitz medium supplemented with 20% serum was limited by the depletion of glutamine. A simple modification of the Leibovitz medium by increasing the concentrations of glutamine, lysine, isoleucine, leucine, sodium pyruvate, galactose, and vitamins resulted in over 10o% increase in cell growth yield. The total myeloma protein produced by the cells was increased by approximately 90% in modified Leibovitz media. Analysis of spent culture media for 19 amino acids showed that the concentrations of 8 amino acids were reduced; those of 5 amino acids were increased and the other 6 did not change significantly.
Classification of loops of lampbrush chromosomes according to the arrangement of transcriptional complexes
Authors: Scheer U. , Franke W. W. , Trendelenburg M. F. , Spring H.
Published: April 2021

The arrangement of transcriptional units in the loops of lampbrush chromosomes from oocyte nuclei of urodele amphibia and from primary nuclei of the green alga Acetabularia have been studied in the electron microscope using spread preparations. Loops with different patterns of arrangement of matrix units (i.e. to a first approximation, transcriptional units) can be distinguished: (i) loops consisting of one active transcriptional unit; (ii) loops containing one active transcriptional unit plus additional fibril-free, i.e. apparently untranscribed, intercepts that may include ‘spacer’ regions; (iii) loops containing two or more transcriptional units arranged in identical or changing polarities, with or without interspersed apparent spacer regions. Morphological details of the transcriptional complexes are described. The observations are not compatible with the concept that one loop reflects one and only one transcriptional unit but, rather, lead to a classification of loop types according to the arrangement of their transcriptional units. We propose that the lampbrush chromosome loop can represent a unit for the coordinate transcription of either one gene or a set of several (different) genes.