bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for incorporation of free-floating mesothelial cells as a mechanism of serosal healing
Authors: Foley-Comer Adam J. , Herrick Sarah E. , Al-Mishlab Talib , Prêle Cecilia M. , Laurent Geoffrey J. , Mutsaers Steven E.
Published: April 2021

Regeneration of the mesothelium is unlike that of other epithelial-like surfaces, as healing does not occur solely by centripetal migration of cells from the wound edge. The mechanism of repair of mesothelium is controversial,but it is widely accepted, without compelling evidence, that pluripotent cells beneath the mesothelium migrate to the surface and differentiate into mesothelial cells. In this study we examined an alternative hypothesis, using in vivo cell-tracking studies, that repair involves implantation,proliferation and incorporation of free-floating mesothelial cells into the regenerating mesothelium. Cultured mesothelial cells, fibroblasts and peritoneal lavage cells were DiI- or PKH26-PCL-labelled and injected into rats immediately following mesothelial injury. Implantation of labelled cells was assessed on mesothelial imprints using confocal microscopy, and cell proliferation was determined by proliferating cell nuclear antigen immunolabelling. Incorporation of labelled cells, assessed by the formation of apical junctional complexes, was shown by confocal imaging of zonula occludens-1 protein. Labelled cultured mesothelial and peritoneal lavage cells, but not cultured fibroblasts, implanted onto the wound surface 3, 5 and 8 days after injury. These cells proliferated and incorporated into the regenerated mesothelium, as demonstrated by nuclear proliferating cell nuclear antigen staining and membrane-localised zonula occludens-1 expression,respectively. Furthermore, immunolocalisation of the mesothelial cell marker HBME-1 demonstrated that the incorporated, labelled lavage-derived cells were mesothelial cells and not macrophages as it had previously been suggested. This study has clearly shown that serosal healing involves implantation,proliferation and incorporation of free-floating mesothelial cells into the regenerating mesothelium.
Essential role of human CDT1 in DNA replication and chromatin licensing
Authors: Rialland Mickael , Sola Francesco , Santocanale Corrado
Published: April 2021

Formation of pre-replicative complexes at origins is an early cell cycle event essential for DNA duplication. A large body of evidence supports the notion that Cdc6 protein, through its interaction with the origin recognition complex, is required for pre-replicative complex assembly by loading minichromosome maintenance proteins onto DNA. In fission yeast and Xenopus, this reaction known as the licensing of chromatin for DNA replication also requires the newly identified Cdt1 protein. We studied the role of hCdt1 protein in the duplication of the human genome by antibody microinjection experiments and analyzed its expression during the cell cycle in human non-transformed cells. We show that hCdt1 is essential for DNA replication in intact human cells, that it executes its function in a window of the cell cycle overlapping with pre-replicative complex formation and that it is necessary for the loading of minichromosome maintenance proteins onto chromatin. Intriguingly, we observed that hCdt1 protein, in contrast to other licensing factors, is already present in serum-deprived G0 arrested cells and its levels increase only marginally upon re-entry in the cell cycle.
The time course and chromosomal localization of recombination-related proteins at meiosis in the mouse are compatible with models that can resolve the early DNA-DNA interactions without reciprocal recombination
Authors: Moens Peter B. , Kolas Nadine K. , Tarsounas Madalena , Marcon Edyta , Cohen Paula E. , Spyropoulos Barbara
Published: April 2021

During mouse meiosis, the early prophase RAD51/DMC1 recombination protein sites, which are associated with the chromosome cores and which serve as markers for ongoing DNA-DNA interactions, are in ten-fold excess of the eventual reciprocal recombinant events. Most, if not all, of these early interactions are eliminated as prophase progresses. The manner in which these sites are eliminated is the focus of this investigation. We report that these sites acquire replication protein A, RPA and the Escherichia coliMUTS homologue, MSH4p, and somewhat later the Bloom helicase, BLM, while simultaneously losing the RAD51/DMC1 component. Eventually the RPA component is also lost and BLM sites remain. At that time, the MUTL homologue, MLH1p,which is essential for reciprocal recombination in the mouse, appears in numbers and locations that correspond to the distribution of reciprocal recombination events. However, the MLH1 foci do not appear to coincide with the remaining BLM sites. The MLH1p is specifically localized to electron-microscope-defined recombination nodules. We consider the possibility that the homology-search RAD51/DMC1 complexes are involved in homologous chromosome synapsis but that most of these early DNA-DNA interactions are later resolved by the anti-recombination RPA/MSH4/BLM-topoisomerase complex,thereby preventing the formation of superfluous reciprocal recombinant events.
Remodeling the intercalated disc leads to cardiomyopathy in mice misexpressing cadherins in the heart
Authors: Ferreira-Cornwell M. Celeste , Luo Yang , Narula Navneet , Lenox Jennifer M. , Lieberman Melanie , Radice Glenn L.
Published: April 2021

The contractile force of the cardiomyocyte is transmitted through the adherens junction, a component of the intercalated disc, enabling the myocardium to function as a syncytium. The cadherin family of cell adhesion receptors, located in the adherens junction, interact homophilically to mediate strong cell-cell adhesion. Ectopic expression of cadherins is associated with changes in tumor cell behavior and pathology. To examine the effect of cadherin specificity on cardiac structure and function, we expressed either the epithelial cadherin, E-cadherin, or N-cadherin in the heart of transgenic mice. E-cadherin was localized to the intercalated disc structure in these animals similar to endogenous N-cadherin. Both N- and E-cadherin transgenic animals developed dilated cardiomyopathy. However, misexpression of E-cadherin led to earlier onset and increased mortality compared with N-cadherin mice. A dramatic decrease in connexin 43 was associated with the hypertrophic response in E-cadherin transgenic mice. Myofibril organization appeared normal although, vinculin, which normally localizes to the intercalated disc, was redistributed to the cytoplasm in the E-cadherin transgenic mice. Furthermore, E-cadherin induced cyclin D1, nuclear reduplication, and karyokinesis in the absence of cytokinesis, resulting in myocytes with two closely opposed nuclei. By contrast, N-cadherin overexpressing transgenic mice did not exhibit an increase in cyclin D1,suggesting that E-cadherin may provide a specific growth signal to the myocyte. This study demonstrates that modulation of cadherin-mediated adhesion can lead to dilated cardiomyopathy and that E-cadherin can stimulate DNA replication in myocytes normally withdrawn from the cell cycle.
Novel syntaxin gene sequences fromGiardia, Trypanosomaand algae: implications for the ancient evolution of the eukaryotic endomembrane system
Authors: Dacks Joel B. , Doolittle W. Ford
Published: April 2021

SNAP receptors or SNARES are crucial components of the intracellular membrane system of eukaryotes. The syntaxin family of SNAREs have been shown to have roles in neurotransmission, vesicular transport, membrane fusion and even internal membrane compartment reconstruction. While syntaxins and SNAREs in general have been well characterized in mammalian and yeast models, little is known about their overall distribution across eukaryotic diversity or about the evolution of the syntaxin gene family. By combining bioinformatic,molecular biological and phylogenetic approaches, we demonstrate that various syntaxin homologs are not only present in `eukaryotic crown taxa' but across a wide range of eukaryotic lineages. The alignment of evolutionarily diverse syntaxin paralogs shows that an isoleucine residue critical to nSec1—syntaxin complex formation and the characteristic syntaxin glutamine residue are nearly universally conserved, implying a general functional importance for these residues. Other identified functional residues involved in botulism toxicity and calcium-binding-protein interactions are also compared. The presence of Golgi-related syntaxins in the intestinal parasite Giardia intestinalis provides further evidence for a cryptic Golgi in this `adictyosomal' taxon, and another likely case of secondary reduction in this parasite. The phylogeny of syntaxins shows a number of nested duplications, including a case of parallel evolution in the plasma membrane-associated syntaxins, and ancestral duplications in the other syntaxin paralogs. These speak to ancient events in the evolution of the syntaxin system and emphasize the universal role of the syntaxins in the eukaryotic intracellular compartment system.
Stem cell factor activates telomerase in mouse mitotic spermatogonia and in primordial germ cells
Authors: Dolci Susanna , Levati Lauretta , Pellegrini Manuela , Faraoni Isabella , Graziani Grazia , Di Carlo Anna , Geremia Raffaele
Published: April 2021

The discovery of sterility in the descendants of telomerasenull mutant mice, owing to the lack of spermatogonia proliferation, has drawn attention to the role of telomerase activity in mouse spermatogenesis. Since spermatogonia proliferation is under Kitl control, we explored its possible role in the regulation of telomerase activity. We show that Kitl induces telomerase activity in mitotic spermatogonia and increases the mRNA levels of both the catalytic subunit form and the telomerase RNA template. The increase of telomerase activity by Kitl is blocked by the presence of the PI3K inhibitor LY294002. Kit-positive proliferating male primordial germ cells (PGCs) show low levels of telomerase activity, but they increase telomerase activity upon Kitl stimulation. Diplotene-arrested growing oocytes that reexpress Kit do not increase telomerase activity upon Kitl stimulation. Our data suggest that the induction of telomerase by Kitl may contribute to the self-renewing potential of male germ cells and of PGCs.
Membrane topology and mitochondrial targeting of mitofusins, ubiquitous mammalian homologs of the transmembrane GTPase Fzo
Authors: Rojo Manuel , Legros Frédéric , Chateau Danielle , Lombès Anne
Published: April 2021

Two human Fzo-homologs, mitofusins Mfn1 and Mfn2, are shown by RT-PCR and western blot to be ubiquitous mitochondrial proteins. Protease digestion experiments reveal that Mfn2 is an outer membrane protein with N-terminal and C-terminal domains exposed towards the cytosol. The transmembrane and C-terminal domains of Mfn2 (Mfn2-TMCT) are targeted to mitochondria and deletion of these domains leads to the cytosolic localization of truncated Mfn2 (Mfn2-NT). Mfn2 is targeted to the endoplasmic reticulum or to mitochondria when the C-terminal domain is replaced by short stretches of neutral/hydrophobic (Mfn2-IYFFT) or polar/basic (Mfn2-RRD) amino acids. The coiled-coil domains of Mfn2, upstream and downstream of the transmembrane domain, are also important for mitochondrial targeting: Mfn2-mutants deleted of any of its coiled-coil domains are only partially targeted to mitochondria and significant protein amounts remain cytosolic. We show that these coiled-coil domains interact with each other: mistargeted Mfn2-NT or Mfn2-IYFFT localize to mitochondria if co-expressed with Mfn2-TMCT. This relocalization is abolished when the coiled-coil domain is deleted in any of the co-transfected molecules. We also found that Mfn2 can cluster active mitochondria in the perinuclear region independently of the cytoskeleton,bring mitochondrial membranes into close contact and modify mitochondrial structure, without disturbing the integrity of the inner and outer membrane.
Trafficking of tail-anchored proteins: transport from the endoplasmic reticulum to the plasma membrane and sorting between surface domains in polarised epithelial cells
Authors: Bulbarelli Alessandra , Sprocati Teresa , Barberi Massimo , Pedrazzini Emanuela , Borgese Nica
Published: April 2021

Tail-anchored (TA) proteins, which are defined by an N-terminal cytosolic region and a C-terminal transmembrane domain (TMD), provide useful models for studying the role of the TMD in sorting within the exo-endocytic system. Previous work has shown that a short TMD is required to keep ER-resident TA proteins from escaping to downstream compartments of the secretory pathway. To investigate the role of the TMD in TA protein sorting, we used model constructs, which consisted of GFP linked at its C-terminus to the tail region of cytochrome b(5) with TMDs of differing length or hydrophobicity. Expression of these constructs in CV-1 cells demonstrated that the feature determining exit from the ER is hydrophobicity and that if exit occurs, at least a part of the protein reaches the cell surface. To investigate which pathway to the surface is followed by plasma-membrane-directed TA constructs, we expressed the TA constructs in polarised Madin Darby Canine Kidney (MDCK) cells. The constructs with 22 and 25 residue TMDs were localised basolaterally, but addition at the C-terminus of a 20-residue peptide containing an N-glycosylation site resulted in glycosylation-dependent relocation of∼50% of the protein to the apical surface. This result suggests that TA proteins may reach the basolateral surface without a signal or that our constructs contain a weak basolateral determinant that is recessive to the apical information carried by the glycan. To assess the effect of the TMDs of endogenous TA proteins, GFP was linked to the tails of syntaxin 3 and 4, which localise to the apical and basolateral surface, respectively, of MDCK cells. The two GFP fusion proteins showed a different surface distribution, which is consistent with a role for the two syntaxin TMDs in polarised sorting.
Nuclear localization of phosphatidylinositol 4-kinase β
Authors: de Graaf Petra , Klapisz Elsa E. , Schulz Thomas K. F. , Cremers Alfons F. M. , Verkleij Arie J. , van Bergen en Henegouwen Paul M. P.
Published: April 2021

Whereas most phosphatidylinositol 4-kinase (PtdIns 4-kinase) activity is localized in the cytoplasm, PtdIns 4-kinase activity has also been detected in membranedepleted nuclei of rat liver and mouse NIH 3T3 cells. Here we have characterized the PtdIns 4-kinase that is present in nuclei from NIH 3T3 cells. Both type II and type III PtdIns 4-kinase activity were observed in the detergent-insoluble fraction of NIH 3T3 cells. Dissection of this fraction into cytoplasmic actin filaments and nuclear lamina-pore complexes revealed that the actin filament fraction contains solely type II PtdIns 4-kinase,whereas lamina-pore complexes contain type III PtdIns 4-kinase activity. Using specific antibodies, the nuclear PtdIns 4-kinase was identified as PtdIns 4-kinase β. Inhibition of nuclear export by leptomycin B resulted in an accumulation of PtdIns 4-kinase β in the nucleus. These data demonstrate that PtdIns 4-kinase β is present in the nuclei of NIH 3T3 fibroblasts,suggesting a specific function for this kinase in nuclear processes.
Direct binding of NuMA to tubulin is mediated by a novel sequence motif in the tail domain that bundles and stabilizes microtubules
Authors: Haren Laurence , Merdes Andreas
Published: April 2021

In mitosis, NuMA localises to spindle poles where it contributes to the formation and maintenance of focussed microtubule arrays. Previous work has shown that NuMA is transported to the poles by dynein and dynactin. So far, it is unclear how NuMA accumulates at the spindle poles following transport and how it remains associated throughout mitosis. We show here that NuMA can bind to microtubules independently of dynein/dynactin. We characterise a 100-residue domain located within the C-terminal tail of NuMA that mediates a direct interaction with tubulin in vitro and that is necessary for NuMA association with tubulin in vivo. Moreover, this domain induces bundling and stabilisation of microtubules when expressed in cultured cells and leads to formation of abnormal mitotic spindles with increased microtubule asters or multiple poles. Our results suggest that NuMA organises the poles by stable crosslinking of the microtubule fibers.
The novel HECT-type ubiquitin-protein ligase Pub2p shares partially overlapping function with Pub1p inSchizosaccharomyces pombe
Authors: Tamai Katsuyuki K. , Shimoda Chikashi
Published: April 2021

The fission yeast Schizosaccharomyces pombe has three putative ubiquitin-protein ligases of the Nedd4/Rsp5 family, named Pub1p, Pub2p and Pub3p. Pub1p has been reported to be involved in cell cycle regulation and proliferation under acidic pH conditions. Here we characterize Pub2p, which contains a conserved HECT domain and a WW domain but lacks a C2 domain. Transcription of the pub2+ gene was constitutive and further enhanced by nitrogen starvation. A pub2-null mutation gave no remarkable phenotypes, but intensified temperature sensitivity in a pub1Δ background. Moderately overexpressed pub2+ suppressed the temperature sensitivity of pub1Δ cells, which suggests that the function of Pub2p overlaps with that of Pub1p. Overexpression of pub2+ by a strong nmt1 promoter in wild-type strains caused growth arrest and cell elongation, probably owing to defects in G2 progression or the G2/M transition. Unlike Pub1p, however, overexpression of Pub2p did not reduce the levels of Cdc25p. Pub2-GFP was found throughout the cell, especially at the cell surface in the polar regions. Pub2p contains a conserved cysteine residue(Cys639) in its putative catalytic HECT domain that can be thiol-ubiquitinated. Substitution of Cys639 by alanine (Pub2CA) caused a functional defect, because growth arrest and cell elongation were not induced by overexpression of Pub2CA. A chimeric Pub1 protein, in which the HECT domain was replaced by the Pub2 HECT domain, completely suppressed the temperature sensitivity of pub1Δ cells, suggesting that the HECT domain of Pub2p has the catalytic activity of a ubiquitin ligase. We conclude that Pub2p is a HECT-type ubiquitin-protein ligase that shares partially overlapping function with Pub1p.
Calcium signalling in tissue: diversity and domain-specific integration of individual cell response in salivary glands
Authors: Segawa Akihisa , Takemura Haruo , Yamashina Shohei
Published: April 2021

Organ function requires coordinated multicellular activities, which may require proper control of cell signalling dynamics at the supracellular level. By using high-speed confocal microscopy, we studied how calcium signalling is organised in the dissociated rat parotid gland. Salivary gland function is accomplished primarily by the compartmentalized epithelial domains, acini and ducts, the former involved in the production of primary saliva and the latter involved in its modification. Upon muscarinic stimulation with carbachol, both domains showed an increase in intracellular free calcium concentration([Ca2+]i) with distinctive spatiotemporal kinetics, as indicated by the fluo-3 fluorescence. Acini responded initially, and the ducts followed with a time lag of more than 0.3 second. Cells comprising an acinus responded synchronously, whereas those in the ducts responded heterogeneously with respect to the latency period, magnitude of response and the requirement of extracellular calcium to raise [Ca2+]i. ATP also elicited a non-synchronous [Ca2+]i response in the duct domain, under a pattern different from that of carbachol. The synchronous oscillations seen in the acinar domain were made asynchronous by octanol, an agent known to inhibit gap-junction function. Accordingly, a gap junction component, connexin 32, was immunolocalised predominantly between the acinar cells. Moreover, expression of the type 2 inositol (1,4,5)-trisphosphate receptor[Ins(1,4,5)P3R] was homogeneous in the acinar domain but heterogeneous in the duct domain. Together, these data suggest that the calcium signalling system in salivary glands is constructed specifically according to the tissue architecture.
Visualisation of the actin cytoskeleton by cryo-electron microscopy
Authors: Resch Guenter P. , Goldie Kenneth N. , Krebs Angelika , Hoenger Andreas , Small J. Victor
Published: April 2021

An understanding of the mechanisms driving cell motility requires clarification of the structural organisation of actin filament arrays in the regions of cell protrusion termed lamellipodia. Currently, there is a lack of consensus on lamellipodia organisation stemming from the application of alternative procedures for ultrastructural visualisation of cytoskeleton networks. In this study, we show that cryo-electron microscopy of extracted cytoskeletons embedded in a thin layer of vitreous ice can reveal the organisation of cytoskeletal elements at high resolution. Since this method involves no dehydration, drying and contrasting steps that can potentially introduce subtle distortions of filament order and interactions, its application opens the way to resolving the controversial details of lamellipodia architecture.
DdNek2, the first non-vertebrate homologue of human Nek2, is involved in the formation of microtubule-organizing centers
Authors: Gräf Ralph
Published: April 2021

Dictyostelium Nek2 (DdNek2) is the first structural and functional non-vertebrate homologue of human Nek2, a NIMA-related serine/threonine kinase required for centrosome splitting in early mitosis. DdNek2 shares 43% overall amino-acid identity with its human counterpart and 54% identity within the catalytic domain. Both proteins can be subdivided in an N-terminal catalytic domain, a leucine zipper and a C-terminal domain. Kinase assays with bacterially expressed DdNek2 and C-terminal deletion mutants revealed that catalytic activity requires the presence of the leucine zipper and that autophosphorylation occurs at the C-terminus. Microscopic analyses with DdNek2 antibodies and expression of a GFP-DdNek2 fusion protein in Dictyostelium showed that DdNek2 is a permanent centrosomal resident and suggested that it is a component of the centrosomal core. The GFP-DdNek2-overexpressing mutants frequently exhibit supernumerary microtubule-organizing centers (MTOCs). This phenotype did not require catalytic activity because it was also observed in cells expressing inactive GFP-K33R. However, it was shown to be caused by overexpression of the C-terminal domain since it also occurred in GFP-mutants expressing only the C-terminus or a leucine zipper/C-terminus construct but not in those mutants expressing only the catalytic domain or a catalytic domain/leucine zipper construct. These results suggest that DdNek2 is involved in the formation of MTOCs. Furthermore, the localization of the GFP-fusion proteins revealed two independent centrosomal targeting domains of DdNek2, one within the catalytic or leucine zipper domain and one in the C-terminal domain.
Synthesis and secretion of growth hormone in the rat anterior pituitary: I. the intracellular pathway, its time course and energy requirements
Authors: Howell S. L. , Whitfield Margaret
Published: April 2021

The intracellular processes involved in synthesis, transport and storage of newly synthesized proteins in the rat somatotroph, together with their time course and metabolic requirements, have been investigated in a quantitative electron-microscopic radioautography study of the tissue following pulse labelling with tritium-labelled amino acids and chase incubations in various conditions. Proteins are synthesized initially on the rough-surfaced elements of the endoplasmic reticulum and are transported within 10 min after their synthesis to transitional areas between the rough-surfaced endoplasmic reticulum and Golgi complex. Transfer to the Golgi lamellae is achieved, probably via transfer vesicles, within about 60 min after synthesis, while formation of mature storage granules occurs within 2 h following protein synthesis. Further experiments utilizing cycloheximide or ouabain during the chase incubations showed that the intracellular transport of newly synthesized protein and its time course are not significantly affected by inhibitors of protein synthesis, or by inhibition of sodium-potassium dependent ATPase by ouabain. Inhibitors of oxidative phosphorylation (250 μM 2,4-dinitrophenol) or of respiration (10 μM antimycin A) markedly reduced intracellular ATP levels and inhibited the intracellular transport processes. The requirement for ATP appeared to be operative at 2 stages: in the movement of transfer vesicles to the Golgi complex and in the formation of storage granules; possible roles of ATP in these processes are discussed.
The effect of spindle inhibitors applied before meiosis on meiotic chromosome pairing
Authors: Dover G. A. , Riley R.
Published: April 2021

Injection of 0·5 % colchicine into immature tillers of genotypes of Triticum aestivum, T. aestivum × Aegilops mutica and T. aestivum x Secale cereale hybrids induces asynapsis at first meiotic metaphase irrespective of the homologous or homoeologous nature of the potential pairing chromosomes. The induction of asynapsis occurs at a time during and immediately following the last premeiotic mitosis of pollen mother cells. No disruption of synapsis and chiasma formation occurs in anthers having pollen mother cells originally at leptotene or immediately prior to leptotene when cultured in White’s medium plus colchicine. Tetraploid and octaploid pollen mother cells resulting from the disruption of premeiotic spindles by colchicine show pairing of chromosomes only in bivalents, in genotypes normally having a degree of multivalent pairing configurations. The induction of multipolar mitotic spindles with 0·01 % colchicine results in the development of pollen mother cell mosaics with different numbers of chromosomes. Such cells show high levels of chromosome pairing, including multivalents, in some genotypes that normally have very little chromosome pairing. The injection of 0·5 % chloral hydrate during the last premeiotic mitosis of the archesporium causes no disturbances of meiotic pairing. The results are discussed with reference to the hypothesis that the control mechanism of meiotic chromosome pairing involves centromeric microtubules of the spindle (not affected by chloral hydrate) that are responsible for the positional adjustment, during the last mitotic anaphase, of potential pairing partners.
The turnover of collagen in fibroblast cultures
Authors: Steinberg J.
Published: April 2021

Collagen turnover was studied in mouse fibroblast cultures (3T6) by radioactive labelling and compartmental analyses. The incorporation of [14C]proline into protein during continuous labelling rapidly reached a maximum value which was directly proportional to the medium specific activity. Radioactivity appeared more slowly in hydroxyproline, and gradually accumulated as cultures became enriched in collagen and its breakdown products. In relation to total new protein synthesis, the proportional synthesis of collagen, as measured by the formation of [14C]hydroxyproline, was less in logarithmically growing than in stationary-phase cultures, and little was deposited in the cell layer.

Newly synthesized hydroxyproline was consistently present in all growth media. In stationary-phase cultures, media contained as much as 60 % of the total [14C]hydroxyproline in a form soluble in 0·5 M perchloric acid. Gel filtration chromatography confirmed that this was pre-dominantly free hydroxyproline, only 30 % appearing in small peptides whose degree of hydroxylation suggested their origin from larger collagen molecules. This acid-soluble compartment was taken as a convenient index of collagenolysis, which proved to be significant in both growth states, but was proportionately more important throughout logarithmic growth.

Reincubation of prelabelled cultures in fresh medium containing an excess of non-radioactive prohne (‘chase’ medium) was followed by the degradative loss of labelled cell layer protein. The released radioactivity could be quantitatively recovered in the growth medium for periods up to 6 days; the rate of its appearance was little influenced by the frequency of feeding. Despite extensive dilution of the proline precursor-pool specific activity, synthesis of [14C]-hydroxyproline continued in all chase cultures. The increment appeared largely as collagen breakdown products in the growth medium, and probably arose from 2 principal sources: (1) recently deposited collagen, and (2) the hydroxylation of peptidyl-[14C]proline residues in protocollagen. The balance between these contributions seemed to be dependent upon the extent to which ‘ageing’ of the cell layer collagen had occurred prior to initiating the chase. Radioactive hydroxyproline was rapidly lost from briefly prelabelled cell layers, but was well retained in a macromolecular form when the initial labelling period was sufficiently prolonged. It is proposed that the endogenous collagen-degradative apparatus attacks both young collagen and its polypeptide precursor, but that as the lability of the former substrate rapidly declines, enzyme activity continues to operate on protocollagen to yield [14C]hydroxyproline-containing breakdown products which gradually diminish as the latter substrate pool is exhausted.
The analysis of malignancy by cell fusion: IV. hybrids between tumour cells and a malignant l cell derivative
Authors: Wiener F. , Klein G. , Harris H.
Published: April 2021

Previous experiments showed that when a range of different highly malignant mouse tumour cells were fused with L cell derivatives of low tumorigenicity, the resulting hybrid cells, so long as they retained something close to the complete chromosome sets of both parent cells, had little or no ability to grow progressively in vivo. Tumours arising from the injection of such hybrids were produced not by progressive growth of the cells injected, but by selective overgrowth of cells from which certain specific, but as yet unidentified, chromosomes had been eliminated. In the present experiments the same malignant mouse tumour cells were fused with a highly malignant L cell derivative selected from the wild type cell population by passage through the animal. In all cases the resulting hybrid cells were found to be highly malignant; and the chromosome constitutions of the tumours arising from the injection of these hybrids were not significantly different from those of the cells injected. These findings confirm the interpretation given to the previous work: the L cell derivatives of low tumorigenicity contribute to the hybrid cells some factor, linked to specific chromosomes, that suppresses the malignant character of the tumour cell, and this suppression is removed, with consequent reappearance of the malignant phenotype, when certain chromosomes are eliminated.
Cell multiplication in Tetrahymena cultures after addition of particulate material
Authors: Rasmussen L. , Modeweg-Hansen L.
Published: April 2021

We have studied the effects of adding participate supplements to populations of Tetrahymena pyriformis in 2 % sterile-filtered proteose peptone broth which supports cell multiplication poorly (generation times in excess of 40 h). The tested compounds were: heat-sterilized suspensions of egg albumin, nutritionally inert particles of polystyrene, sulphopropyl and quarternary amino-ethyl substituted dextran (in concentrations of 4, 40 and 400 μg per ml). The particles had approximately the same size, but differed in their electric net charges. Particulate suspensions of 40 μg per ml or more greatly improved cell multiplication rates (generation times about 6 h). It is probable that the effect of the particles is to induce formation of food vacuoles without which cell multiplication and growth is very slow. The contribution of the food vacuole to nutrient uptake in Tetrahymena is discussed.
The porous plates of coniform chloride cells in mayfly larvae: High-resolution analysis and demonstration of solute pathways
Authors: Komnick H. , Stockem W.
Published: April 2021

The porous plates of coniform chloride cells in mayfly larvae were studied by electron microscopy using carbon replicas, whole-mount cuticles and thin sections. The cuticular skeleton of the porous plates is a local differentiation of the epicuticle forming a structure of extremely high organization. It is composed of cylindrical rods, which measure 20 ran in diameter, and are oriented perpendicular to the cuticular surface. They form an overlapping hexagonal pattern and are interconnected by double rows of transverse bars which are arranged in. a complicated but regular fashion. This spongy skeleton defines a continuous space system which may be characterized as composed of triangular pores, the sides of which measure 20 nm. The pores are arranged in a hexagonal pattern, and interconnected via slit-like communications. The porous plates are covered with a porous lamina which possesses 2·5-nm-wide pores in hexagonal arrangement.

Results obtained from negative staining, infiltration with colloidal lanthanum hydroxide and histochemical precipitation of sodium indicate that the porous system provides pathways for solutes across the cuticle.

The 3-dimensional architecture of the porous plates is reconstructed in a model that also includes morphogenetic aspects.
Pattern formation in the blue-green alga,Anabaena: I. basic mechanisms
Authors: Wilcox Michael , Mitchison G. J. , Smith R. J.
Published: April 2021

Filaments of Anabaena have a spaced pattern of differentiated cells called heterocysts, which is maintained as a filament grows by the regular determination of new heterocysts. By following the growth of every cell in a filament, we have identified proheterocysts (prospective heterocysts) at their earliest appearance, and described the sequence of events in the formation of the pattern. The determination of proheterocysts obeys 2 rules: (1) that there are inhibitory zones around pre-existing heterocysts, and (2) that only the smaller daughter of a division can become a heterocyst (all divisions are asymmetrical). There are, however, certain conditions in which these rules are over-ridden, where a pattern consisting of groups of consecutive proheterocysts is seen which resolves into a normal discrete pattern. This process is highly suggestive of interaction between developing cells. We have tested this hypothesis in normal growth conditions by breaking filaments near to early proheterocysts, on the assumption that this will cause a build-up of inhibitory effect of the cell upon itself. It is found that these cells regress, losing their differentiated character and dividing. We therefore propose an interactive model for pattern formation in Anabaena.
capsella embryogenesis: The central cell
Authors: Schulz Patricia , Jensen W. A.
Published: April 2021

The central cell is the binucleate cell of the angiosperm megagametophyte which contains the polar nuclei and participates in double fertilization. The structure of the mature central cell, the fusion of the polar nuclei and the primary endosperm nucleus were studied with the electron microscope. The central cell cytoplasm appears very active and has an extensive ER, many mitochondria, dictyosomes, microbodies, polysomes, chloroplasts with well developed grana and starch and lipid reserves. A single, giant mitochondrion appears in the cytoplasm near the polar nuclei at the time of fertilization, but its origin, fate and function are not known. Cytoplasmic aggregates of dense, granular material are associated with the primary endosperm nucleus and structurally resemble the nucleolus and similar aggregates in the nucleoplasm. It is suggested that these cytoplasmic perinuclear bodies may represent extruded nucleolar material. The central cell cytoplasm does not undergo any notable structural reorganization as a result of fertilization. The relationship of the central cell to the other cells of the mature megagametophyte and its possible role in embryogenesis is discussed.
Prenucleolar bodies of Urechis oocytes
Authors: Das N. K. , Alfert M.
Published: April 2021

The immature oocyte and unfertilized eggs of Urechis caupo, a marine echiuroid worm, contain many nuclear bodies and a main nucleolus. These bodies are similar to prenucleolar bodies of other materials with respect to their origin, structure, cytochemical and biochemical characteristics. They are formed in large numbers during the diffuse diplotene stage, perhaps by an aggregation of nucleolar materials which had been associated with chromosomes. They are fibrillar in structure, made up mainly of some nucleolar proteins, and are inactive in RNA synthesis. Biochemical analyses suggest the possibility that these structures contain a small amount of ribosomal RNA precursors which may have originated from the main nucleolus. An accumulation of prenucleolar bodies in Urechis oocytes may result from the lack of formation of multiple functional nucleoli, such as those found in amphibian oocytes.
Studies on contact inhibition of growth in the mouse fibroblast, 3t3: I. changes in cell size and composition during ‘unrestricted’ growth
Authors: Meisler A. I.
Published: April 2021

Growth of the contact-inhibited 3T3 and its SV40-transformed derivative, 3T3T is unbalanced. Despite obvious exponential increase in cell number, cell volume, RNA and protein show a marked decrease with increasing population density in both cell types, greater in 3T3 than 3T3T. Daily medium change does not correct this growth pattern but merely seems to equalize initial growth rates of the 2 cell types and prolong the period during which the cell number continues to increase. The evidence suggests that the lag period that ensues following plating of the cells is a period of intense metabolic activity during which a large proportion of cellular protein and RNA is synthesized in preparation for exponential growth.
Studies on contact inhibition of growth in the mouse fibroblast, 3t3: II. effects of amino acid deprivation and serum on growth rate
Authors: Meisler A. I.
Published: April 2021

The effect of amino acid concentration on the initial growth rate of the contact-inhibited mouse fibroblasts 3T3 and its SV40-transformed derivative 3T3T has been studied. In medium in which the serum had been passed through a Sephadex column, and the concentration of amino acids lowered 1000-fold, one acid at a time, the growth rate constants were computed. For most of the 16 amino acids studied, the initial growth rate of 3T3 decreased more profoundly than that of 3T3T. The presence in serum of a factor which stimulates amino acid uptake and growth rate of 3T3T is described.
The localization of trypsin in cultured mammalian cells
Authors: Hodges Gisele M. , Livingston D. C. , Franks L. M.
Published: April 2021

The localization of trypsin in HeLa and CBM 17 baby mouse kidney cells was studied using fluorescent and electron-microscope autoradiographical techniques. Intracellular uptake of trypsin, as well as cell surface localization, was demonstrated by the use of direct FITC- and tritium acetylated-labelled trypsin and immunoreactive procedures. Intracellular penetration of the enzyme was temperature dependent and while evident at 37 and 25 °C was negligible at 4 °C. Higher proteolytic activity could be demonstrated in the supernatants from disrupted trypsin-treated cells than in supernatants from disrupted PBS-treated cultures. Treatment of trypsin with serum, whilst depressing enzyme protease activity, did not modify intracellular uptake of the enzyme and intracellular localization of trypsin persisted in cultured cells for up to about 48 h. The results, while not accounting for the primary event in cell alteration following exposure to trypsin, clearly suggest that consideration must be given to the fact that intracellular penetration of the enzyme may affect certain intrinsic processes of the cell.
The effects of fasting and feeding on the protein synthesis rate and polyribosomal profile of frog pepsinogenic cells
Authors: Van Venrooij W. J. , Poort C. , Geuze J. J.
Published: April 2021

1. The rate of [14C]leucine incorporation into the protein of frog pepsinogenic cells was measured in both fasted and fed animals. The incorporation rate increases after feeding, becoming maximal after 2 h. 2. In the oesophagi of fasted frogs, only monoribosomes and dimers are present. The absence of polyribosomes is not due to autolysis in the homogenate. After feeding, heavy polyribosomal aggregates appear. 3. It is concluded that feeding stimulates protein synthesis in frog pepsinogenic cells.
Dissecting the PRSS37 interactome and potential mechanisms leading to ADAM3 loss in PRSS37-null sperm
Authors: Xiong Wenfeng , Shen Chunling , Li Chaojie , Zhang Xiaohong , Ge Haoyang , Tang Lingyun , Shen Yan , Lu Shunyuan , Zhang Hongxin , Han Mi , Zhang Aijun , Wang Jinjin , Wu Youbing , Fei Jian , Wang Zhugang
Published: April 2021

A disintegrin and metalloproteinase 3 (ADAM3) is a sperm membrane protein critical for sperm migration from the uterus into the oviduct and sperm-egg binding in mice. Disruption of PRSS37 results in male infertility concurrent with the absence of mature ADAM3 from cauda epididymal sperm. However, how PRSS37 modulates ADAM3 maturation remains largely unclear. Here, we determine the PRSS37 interactome by GFP immunoprecipitation coupled with mass spectrometry in PRSS37-EGFP knock-in mice. Three molecular chaperones (CLGN, CALR3 and PDILT) and three ADAM proteins (ADAM2, ADAM6B and ADAM4) were identified to be interacting with PRSS37. Coincidently, five of them (except ADAM4) have been reported to interact with ADAM3 precursor and regulate its maturation. We further demonstrated that PRSS37 also interacts directly with ADAM3 precursor and its deficiency impedes the association between PDILT and ADAM3. This could contribute to improper translocation of ADAM3 to the germ cell surface, leading to ADAM3 loss in PRSS37-null mature sperm. The understanding of the maturation mechanisms of pivotal sperm plasma membrane proteins will pave the way toward novel strategies for contraception and the treatment of unexplained male infertility.
On the Physiology of Amoeboid Movement
Authors: Pantin C. F. A.
Published: April 2021

It was shown in the first paper of this series 15 that amoeboid activity was affected by certain changes in the conditions of the medium in the same way as certain other forms of contractility. This suggested that some fundamental mechanism of contractility was similar in all these cases.

Like the majority of biological processes, contractility is affected in a characteristic manner by temperature, and if there really is a fundamental similarity between amoeboid and other forms of contractility, the effect of temperature should be similar in both cases.
Skin Grafts in the Lizard, Anolis Carolinensis, Cuv
Authors: May Raoul M.
Published: April 2021

Autotransplants of pigmented (melanophore-bearing) skin on a white region, and of white skin on a pigmented region both exhibit a strict specificity. There is no migration of the melanophores in any case of transplantation. Transplants of pigmented skin lose their colour-changing power immediately on being completely disconnected from neighbouring tissues, and regain it slowly as nerves regenerate. The melanophores are supplied largely by nerves which reach them directly through the underlying tissues. If these nerves are cut from a given area of skin, without separating the area from the surrounding skin, it at first loses the power of colour change, but the neighbouring integument appears able, after a delay of a few days, to control the denervated area. Areas of melanophore-bearing skin, isolated by incision from surrounding skin, but with their subcutaneous connections intact, can act normally with the rest of the animal in the colour-changes. Adrenalin, in producing the green state in pigmented grafts which have regained the colour-changing function, acts as it does upon normal integument. The action is indirectly through the sympathetic nervous system, and not directly upon the melanophores. Homoiotransplants, either pigmented or white, are absorbed. The absorption occurs after the grafts have well healed in place. The scutes disappear, leaving the area covered by thin scaleless integument. New scutes regenerate wherever there are areas barren of them. Scute regeneration progresses centripetally, suggesting some formative influence exerted by the surrounding normal integument.
The Permeability and Haemolytic Action of the Fatty Acids and Some of their Halogen Derivatives
Authors: Bodansky M.
Published: April 2021

A critical discussion of the methods of studying the permeability of erythrocytes is contained in a recent review by Jacobs (1). In this review the procedures based upon the osmotic volume changes are considered and the unique advantages of the haemolysis method are pointed out in particular. By this method, the behaviour of enormous numbers of cells is made easily visible to the naked eye, through mere changes in the turbidity of the cell suspension. This procedure may therefore be employed as a gross statistical method. Appropriate modifications and improvements in the technique, depending on the nature of the problem, make possible fairly quantitative results. A second advantage of the method is its great delicacy. A third is that by means of certain refinements (2) it may be adapted to experiments whose total duration is only a few seconds.
The Vapour Pressure of the Blood of the Edible Snail
Authors: Kamada Takeo
Published: April 2021

It is an interesting fact that the water content of the edible snail, Helix pomatia, varies with the physiological condition of the animal. Duval (1930) showed that the osmotic pressure of the blood varied from Δ = − 0·37° to ‒ 0·43° in hibernating animals, and from — 0-30° to — 0-40° in active summer animals. Cardot and Troussier (1929) found that the blood volume was considerably greater in active than in hibernating animals, and Brand (1931) showed the same to be true of the water content of the whole body. In the present paper, the observations of Duval (1930) on the osmotic pressure of the blood are repeated, using the very accurate thermo-electric method described by A. V. Hill (1930 a).
The Energy Sources in Ontogenesis
Authors: Needham Joseph
Published: April 2021

The study of the respiratory quotient during embryonic development is of particular interest and importance in view of the body of evidence which has accumulated indicating that the characteristic source of energy changes with time as animal embryos develop from fertilised egg cell to mobile self-supporting organism. This evidence (brought together by Needham (24)) points to an association of carbohydrate catabolism with the earliest stages of development, and to a later appearance of protein and fat catabolism successively. The respiratory quotients which have been obtained in the past on embryonic material form, indeed, a part of this evidence, but a discussion of them will be deferred until the experimental data of the present communication have been dealt with. Up to the present no respiratory measurements of any kind have been made on crustacean embryos, although many of them appear to offer an extremely attractive material to the general physiologist. The eggs of many decapod Crustacea are carried by the females for the whole of their incubation period, and it is therefore only necessary to collect a sufficient number of individual adults, in order to obtain masses of eggs in all developmental stages; and these masses, moreover, will be quite uniform embryologically, since the eggs in them were all fertilised at the same time.