bluuebunny/crossref_metadata_2025 huggingface dataset:
Microtubule dynamic instability: Numerical simulation of microtubule transition properties using a lateral cap model
Authors: Bayley Peter M. , Schilstra Maria J. , Martin Stephen R.
Published: April 2021

We present a numerical formulation for the dynamic instability of microtubules involving the stabilisation of growing microtubules by a single layer of tubulin-GTP, with GTP hydrolysis effectively coupled to tubulin-GTP addition. This Lateral Cap model provides a readily visualised, working mechanism for the co-existence and interconversion of growing and shrinking microtubules.

This class of model is specified in terms of a hydrolysis rule, whereby the addition of tubulin-GTP causes hydrolysis of GTP on a previously terminal tubulin-GTP molecule as it becomes incorporated into the microtubule lattice. A specific formulation is illustrated, though this is not unique. A limited set of parameters defines the kinetics and affinity for tubulin-GTP at the binding sites at a given end of the microtubule. The rate constants are a function of the nucleotide composition of the binding site, principally comprising the two tubulin molecules, which interact laterally and longitudinally with the incoming tubulin-GTP molecule.

The Lateral Cap formulation demonstrates that a single terminal layer of tubulin-GTP is sufficient to reproduce the apparently complex behaviour of a dynamic population of microtubules. It differs significantly from the fluctuating tubulin-GTP cap model of Chen and Hill (1985). It gives a molecular description to the switching of individual microtubules between growing and shrinking states in terms of the composition of the multi-start terminal layer of the microtubule, and provides a general mechanism for the differential kinetic behaviour at opposite ends of dynamic microtubules. It reproduces the essential features of microtubule length excursions, and predicts detailed characteristics of microtubule dynamics, including the basis of the apparently cooperative nature of the transition behaviour as a function of the concentration of tubulin-GTP. It is readily amenable to further experimental test and refinement.
Laminin interactions with ductal pancreatic adenocarcinoma cells: Identification of laminin- and collagen-binding proteins
Authors: Mai M. , Brune K. , Jacoby B. , Kern H. F. , Mollenhauer J.
Published: April 2021

Laminin promotes the modulation of human pancreatic adenocarcinoma cells from a proliferative to a resting phenotype. This process includes restoration of cell polarity, increase of protein biosynthesis, and increase of glycoprotein secretion. The growth correlates with the amount of laminin coated on the culture dish. Adenocarcinoma cells do not synthesize collagen type I, fibronectin or laminin. Prolonged propagation of the cells on laminin substratum enhances the expression of laminin-binding sites on the cell surface. Laminin binds to cell plasma membrane vesicles with a KB of about 2.2×1010. By affinity chromatography of [35S]methionine-labeled, detergent-extracted, cells on immobilized laminin, a Mr82000 polypeptide could be enriched. By simultaneous chromatography on immobilized collagen type I, a Mr34000 polypeptide was retained. In laminin overlay experiments on transblotted plasma membrane proteins, Mr82000 and 70000 polypeptides were labeled. Affinity chromatography on laminin-Sepharose of tumor cell membranes from cells grown in nude mice tumors retained Mr 100 000, 82000, 70000 and 55000 polypeptides bound to the column. Endoglycosidase F treatment of these proteins reduced the number of higher Mr proteins, leaving a Mr 70 000 polypeptide, together with smaller peptides. Using the enriched binding protein fraction as antigens, monoclonal antibodies (mAbs) were prepared in mice. Two of the mAbs were further analysed; they recognized simultaneously the complete pattern of high and low Mr polypeptides identified to date by the other methods. Antibody 2A1-H7 was capable of inhibiting attachment and spreading of the cells on laminin and collagen I, but not on tissue-culture plastic. These data may indicate a molecular heterogeneity, partly based on diverse glycosylation, responsible for the variations in the molecular weight of the binding proteins. The adaptation processes of the tumor cells during growth on the extracellular matrix may indicate a regulatory function on tumor growth and metastasis in vivo.
Monoclonal antibody against a conjugation-specific nuclear antigen in Paramecium caudatum
Authors: Yanagi Akira , Yamamoto Hiroaki
Published: April 2021

To understand molecular mechanisms controlling the sexual reproduction process (conjugation) in Paramecium caudatum, we have tried to detect conjugation-specific antigens with monoclonal antibodies. We obtained a monoclonal antibody (CSN-1) against an antigen that appears in the nuclei only during conjugation. This nuclear antIgen began to appear both in microand macronuclei at micronuclear stage II or HI early in the conjugation process (4 h after the mating reaction at 25°C). In the macronucleus, the nuclear antigen persisted until the stage of macronuclear fragmentation (about 35 h) and then disappeared before degeneration of the macronuclear fragments. In the micronucleus, the antigen existed until the crescent stage (stage V) of the first meiotic division (8h). The antigen in the micronucleus disappeared after the crescent stage but reappeared again in the eight nuclei produced by the third postzygotic division (25 h). Then it persisted only in the four macronuclear anlagen differentiated from the eight nuclei (about 30 h). When exconjugant cells had undergone two successive postconjugational cell divisions and thus possessed only one new macronucleus as in the vegetative cells, the antigen disappeared completely from the new macronucleus in most cells. These cells without the antigen began to appear about 50 h after the mating reaction. As the antigen is specific to conjugation and localized in nuclei, it may play some important role(s) in the conjugation process.
Poly-N-acetyllactosamine-specific tomato lectin interacts with gastric parietal cells Identification of a tomato-lectin binding 60-90×103/Mr membrane glycoprotein of tubuloveslcles
Authors: Callaghan Judy M. , Toh Ban-Hock , Pettitt Ohn M. , Humphris Danielle C. , Gleeson Paul A.
Published: April 2021

The cytoplasmic tubulovesicular and canalicular membranes of gastric parietal cells are intimately involved in hydrochloric acid secretion. To characterise the glycoproteins of these membranes, we examined a panel of lectins for reactivity with parietal cells in paraffin sections of rat, dog and pig stomach. The poly-N-acetyllactosamine-specific lectin from Lycopersicon esculentum (tomato) and from Solatium tuberosum (potato), and the galactose-specific lectin Ricinus communis agglutinin (RCAt2o), showed strong cytoplasmic binding of parietal cells of all three species, with a pattern indicative of an intracellular membrane network. Binding to parietal cells was confirmed by doublelabelling studies with parietal cell auto-antibodies from patients with autoimmune gastritis. Mucous cells and mucin also bound these lectins strongly. Other gastric cell types did not stain with either tomato or potato lectin, but stained weakly with RCA12O-Electron-microscopic examination of lectin binding sites using biotinylated tomato lectin or RCA120 and streptavidin-gold, revealed specific binding to the luminal face of parietal cell tubulovesicular and canalicular membranes as well as the contents of mucous cell secretory granules.

Tomato lectin and RCA120 reacted by lectin blotting with a major species of apparent molecular weight 60-90×103Mr from rat, dog and pig gastric membranes. A tubulovesicular membrane fraction, enriched 10-fold for K+-dependent phosphatase activity, was also enriched three-fold for tomato lectin binding as assessed by a solid-phase lectin assay. The 60-90K (K=103Mr) component, in 125I-labelled detergent extracts of dog tubulovesicular membranes, bound to an affinity support of tomato lectin-Sepharose and was specifically eluted with N,AT,AT’-triacetylchitotriose. Digestion with N-glycanase collapsed the 60-90K component into a sharp 35K band.

We conclude that: (1) a 60-90K membrane glycoprotein localised on the luminal face of tubulovesi-cles and canaliculi of parietal cells interacts strongly with tomato lectin and RCA120; and (2) the glycoprotein is composed of a 35K core protein glycosylated with N-glycans probably containing poly-N-acetyllactosamine sequences with terminal galactosyl residues. The properties of this 60-90K glycoprotein are identical to a major parietal cell autoantigen recognised by sera of patients with autoimmune gastritis. As this molecule represents the only component other than the 95K catalytic subunit of the H+/K+-ATPase, which has been specifically identified with parietal cell tubulovesi-cles, and as it has a similar sized protein core to that of the β subunit of the Na+/K+-ATPase, we propose that it is the β subunit of the proton pump.
Keratinocyte transglutaminase in human skin and oral mucosa: cytoplasmic localization and uncoupling of differentiation markers
Authors: Ta Bach Mai , Gallagher George T. , Chakravarty Rupa , Rice Robert H.
Published: April 2021

Expression of kératinocyte transglutaminase, a specific differentiation marker, has been examined by immunogold-silver cytochemistry in human epidermis and oral epithelium, and in oral mucosal hyperplasia and neoplasia. Two major findings have been obtained. First, considerable immunoreactivity was evident not only at the plasma membrane (the site of cross-linked envelope formation) but also in the cytoplasm of spinous cells, suggesting a cytoplasmic function for this transglutaminase. Staining at the cell border was seen principally in the granular layer of orthokeratinized epithelium (epidermis, hard palate), the outer spinous cells of ortho- and parakeratinized epithelium and in the suprabasal cells showing squamous differentiation in benign and malignant neoplasms. By contrast, diffuse cytoplasmic staining was observed in the upper spinous layer of the normal epithelium and benign lesions. The cytoplasmic immunoreactivity, which extended nearly to the basal layer in hyperkeratosis of the oral mucosa, was evident in two of three verrucous carcinomas examined. In keeping with their undifferentiated character, invasive nests of squamous cell carcinoma and basaloid epithelium in benign and neoplastic lesions were immunonegative for transglutaminase. The second major finding was that lesions of severe oral epithelial dysplasia, immunonegative for transglutaminase, were capable of expressing involucrin immunoreactivity, indicating an uncoupling of keratinocyte programming. These results suggest that immunogold-silver staining for transglutaminase may be useful in evaluating the degree of differentiation in benign and malignant oral epithelial proliferation.
Role of nuclear protein kinase C in the mitogenic response to platelet-derived growth factor
Authors: Fields Alan P. , Tyler Greg , Kraft Andrew S. , May W. Stratford
Published: April 2021

We have assessed the involvement of nuclear envelope protein phosphorylation in the mitogenic response to platelet-derived growth factor (PDGF) in NIH/3T3 fibroblasts. We find that stimulation of quiescent NIH/3T3 cells with PDGF or with the mitogenic protein kinase C (PKC) activators phorbol 12-myristate 13-acetate (PMA) or bryostatin 1 (bryo) leads to rapid, dose-dependent phosphorylation of several nuclear envelope polypeptides. The predominant nuclear envelope targets for mitogen-induced phosphorylation are immunologically identified as the nuclear envelope lamins. All three lamin species (A, B and C) are phosphorylated in response to PMA or bryo, while lamins A and C are preferentially phosphorylated in response to PDGF. Phosphopeptide mapping and phosphoamino acid analysis indicate that similar serine sites on the lamins are phos phorylated in response to PDGF, PMA and bryo. Both mitogenicity and lamina phosphorylation induced by these mitogens can be inhibited by the selective PKC inhibitor staurosporine at 2 nM. Treatment of quiescent NLH/3T3 cells with PDGF, PMA or bryo leads to rapid translocation of PKC to the nuclear envelope. These data indicate that rapid nuclear events, including translocation of cytosolic PKC to the nuclear membrane and lamina phosphorylation, may play a role in the transduction of the mitogenic signals of PDGF from the cytoplasm to the nucleus in NTH/3T3 fibroblasts.
Binding of human endothelium to Ulex europaeus l-coated Dynabeads: application to the isolation of microvascular endothelium
Authors: Jackson C. J. , Garbett P. K. , Nissen B. , Schrieber L.
Published: April 2021

A major problem encountered when isolating human microvascular endothelium is the presence of contaminating cells such as fibroblasts that rapidly over-grow the endothelial cells. We describe here a simple, rapid technique for purifying endothelial cells derived from the microvasculature of neonatal foreskin and osteoarthritic and rheumatoid arthritic synovium. This technique is based on the selective binding of the lectin Ulex europaeus I (UEA I) to the endothelial cell surface via fucose residues. Initially UEA I was covalently bound to tosyl-activated super-paramagnetic polystyrene beads (Dynabeads) by incubation for 24 h at room temperature. Cells were isolated by extracting microvascular segments from enzyme-treated (trypsin and Pronase) cubes of tissue. The mixed population of cells obtained were purified by incubating them at 4 °C for 10 min with the UEA I-coated Dynabeads. Endothelium bound to the beads whilst contaminating cells were removed by five washes with HBSS using a magnetic particle concentrator. The endothelial cells thus obtained grew to confluence as a cobblestone-like monolayer and expressed von Willebrand factor antigen. The cells were released from the Dynabeads by the competitive binding of fucose (10 min at 4°C). This new method is simple and reproducible and allows pure human microvascular endothelial cells to be cultured within 2 h of obtaining a specimen.
The transition of cells of the fission yeast β-tubulin mutant nda3-311 as seen by freeze-substitution electron microscopy Requirement of functional tubulin for spindle pole body duplication
Authors: Kanbe Tosfflo , Hiraoka Yasushi , Tanaka Kenji , Yanagida Mitsuhiro
Published: April 2021

A previous fluorescence light-microscopic study showed that the fission yeast cold-sensitive β-tubulin mutant nda3-311 was arrested with rod-like condensed chromosomes in a mitotic state at the restrictive temperature. Upon transfer to the permissive temperature, a spindle was formed and the nucleus was divided. In the present study, we employed freeze-substitution electron microscopy to examine the ultrastructure of arrested and released nda3-311 cells. In arrested cells, a single, displaced nucleus was seen with a single spindle pole body. Therefore, spindle pole body duplication seemed to require functional β-tubulin. The nuclear membrane was highly deformed with a leaf-like profile in cross-section, possibly due to an interaction with the rod-like, condensed chromosomes. Upon transfer to the permissive temperature, the spindle pole duplicated and the daughter spindle pole bodies rapidly migrated to the opposite ends of the nucleus, accompanied by the formation of the mitotic spindle. Elongation of the nuclear envelope occurred with concomitant spindle extension, as in a wild-type mitosis. The deformed nuclear membrane became smooth and described a convex curve. The numerous vacuoles that are seen in the arrested cells decreased in number and increased in size. Septation was completed, leaving the two divided nuclei in one half of the cell. Hexagonally arranged microtubules, apparently forming the mitotic spindle, were observed in a cross-section of a cell after return to the permissive conditions.
Variable pathways for developmental changes in composition and organization of microtubules in Physarum polycephalum
Authors: Solnica-Krezel Lilianna , Diggins-Gilicinski Maureen , Burland Timothy G. , Dove William F.
Published: April 2021

The development of uninucleate amoebae into multinucleate plasmodia in myxomycètes is called the amoebal-plasmodial transition (APT). During the APT in Physarum polycephalum the ability to form flagellar axonemes is lost; the astral, open mitosis is replaced by the anastral, closed mitosis; and cyto-skeletal microtubules disappear. These changes are accompanied by alterations in the repertoire of expressed tubulins. Using immunofluorescence microscopy we have studied the timing of loss and accumulation of developmentally regulated tubulin isotypes in relation to other cellular events during the APT. We specifically asked whether changes in the composition of microtubules are correlated with changes in their organization. The plasmodiumspecific ββ2-tubulin can first be detected in microtubules of uninucleate cells after they become committed to plasmodium formation. However, rare cells are observed that exhibit ββ2-tubulin at earlier or only at later stages of development. Amoeba-specific acetylated α3-tubulin disappears gradually during development. Individual cells differ in the timing of loss of this isotype: α3-tubulin is present in themajority of uninucleate cells, in a fraction of binucleate and quadrinucleate cells, and is absent from larger multinucleate cells. Cytoplasmic microtubules in uninucleate cells are organized by a single microtubule-organizing center (MTOC) juxtaposed to the nucleus. Binucleate cells and quadrinucleate cells exhibit variable numbers of MTOCs. Cytoplasmic microtubules persist during the APT until the stage of plasmodia containing at least 100 nuclei. The lack of a strict correlation between the changes in tubulin composition and changes in organization of microtubular structures indicates that accumulation of β2- tubulin and disappearance of α3tubulin isotypes are not sufficient to bring about reorganization of microtubules during development. Individual cells in a developing population differ not only in the succession of accumulation and loss of developmentally regulated tubulins, but also in the sequences of other cellular changes occurring during the APT.
Alteration in expression of smooth muscle α-Actin associated with transformation of rat 3Y1 cells
Authors: Okamoto-Inoue Mitsuse , Taniguchi Shuntchiro , Ano Hiroyuki Sad , Kawano Toyokazu , Kimura Genki , Gabbiani Giulio , Baba Tsuneo
Published: April 2021

Expression of actin was examined in a cultured rat embryonic cell line 3Y1 and transformed cell lines that originated from 3Y1. An α-actin in addition to cytoplasmic α- and γ-actins was detected in 3Y1 by two-dimensional gel electrophoresis. This α-Actin was hardly detected at all in the transformants induced by Rous sarcoma virus, v-H-ras oncogene or adenovirus type 12, while the α-Actin was retained in the transformed cell lines induced by N-methyl-N′-nitro-N-nitrosoguanidine or in SV40, which are cell lines of relatively low malignancy. Western and Northern blot analyses established that this α-actin was a smooth muscle a-isoform. An immunofluorescence study revealed that smooth muscle α-Actin in 3Y1 cells is present in stress fibers. Thus, smooth muscle α-Actin is also a component of actin stress fibers, as α- and γ-actins are in 3Y1 cells. An alteration in the expression of this actin isoform may be related to phenotypical changes accompanying transformation.
The F1 ATP synthetase β-subunit: A major yeast novobiocin binding protein
Authors: Jenkins John R. , Pocklington Michael J. , Orr Elisha
Published: April 2021

Novobiocin affects DNA metabolism in both prokaryotes and eukaryotes, resulting in cell death. Ln prokaryotes, the drug is a specific inhibitor of DNA gyrase, a type II topoisomerase that can be purified on a novobiocin-Sepharose column. The yeast type II topoisomerase is neither the biochemical, nor the genetic target of the antibiotic. We have purified the major yeast novobiocin binding proteins and identified one of them as the β-subunit of the yeast mitochondrial Fi ATP synthetase, a protein highly conserved throughout evolution. The inactivation of this protein might explain the toxic effects of novobiocin on higher eukaryotic cells.
Differential distribution of xyloglucan glycosyl transferases in pea Golgi dictyosomes and secretory vesicles
Authors: Brummell David A. , Camirand Anne , Maclachlan Gordon
Published: April 2021

Rate-zonal centrifugation of pea (Pisum sativum var. Alaska) stem microsomal membranes on a linear Renografin gradient separated Golgi secretory vesicles from dictyosomes. Secretory vesicles possessed high levels of xyloglucan fucosyl transferase activity, which effects the final decoration of stem xyloglucan side-chains, but lacked substantial xyloglucan xylo-syl transferase activity, which is required for the synthesis of the xyloglucan backbone. In contrast, total dictyosomal membranes possessed both fucosyl and xylosyl transferase activities. Isopycnic centrifugation of a homogenized dictyosome-enriched membrane preparation on a shallower Renografin gradient indicated that lighter dictyosomal membranes possessed xylosyl transferase but relatively little fucosyl transferase activity. The bulk of the dictyosomal membranes formed a denser peak in which xylosyl and fucosyl transferase activities codistributed. Thus a differential localization of function in the Golgi apparatus during biosynthesis of xyloglucan is indicated. A tentative mechanism is suggested in which the elaboration of the glucose-xylose backbone is initiated in lighter dictyosomal membranes, backbone synthesis is concluded and fucosylation begun in denser dictyosomal membranes, and fucosylation completed in Golgi secretory vesicles during transport of xyloglucan to the cell wall.
Measurement of intracellular pH in fungal hyphae using BCECF and digital imaging microscopy: Evidence for a primary proton pump in the plasmalemma of a marine fungus
Authors: Davies Julia M. , Brownlee C. , Jennings D. H.
Published: April 2021

The facultative marine fungus, Dendryphiella salina, has the most negative membrane potential yet recorded for a marine organism. The ionic basis for this is thought to be through the action of a primary proton pump, though there exists the possibility of electrogenic pumping of Na+ or Cl−, given the high ambient concentration of these ions. Fluorescence ratio imaging microscopy with the pH-sensitive fluorescent probe 2′,7′-bis-(2-carboxyethyl)-5(and-6) carboxyfluorescein (BCECF) has been used to estimate intracellular pH. Hyphae loaded readily with BCECF after incubation with the acetoxymethyl ester (BCECF/AM). Mean resting intracellular pH (pHi) was 7.3, calculated by comparing 490/450 nm fluorescence ratios with in vivo calibration curvesobtained by pH equilibration using nigericin. Distinct pH compartments could be observed, corresponding to cytoplasmic and smaller vacuolar compartments. Sodium azide reversibly reduced pHi by an average of 0.51 of a pH unit, though the response varied between individual hyphae. Inhibiting the plasmalemma ATPase with orthovanadate also reversibly decreased pHi. The results support the presence of a proton pump in the plasmamembrane. The energetic and evolutionary implications are discussed.
Temporal expression, polar distribution and transition of an epitope domain in the perinuclear theca during mouse spermatogenesis
Authors: Bellvé Anthony R. , Chandrika R. , Barth Alinda
Published: April 2021

A novel domain of epitopes is expressed by a family of high-Mr proteins at the anterior pole of the germ cell nucleus during spermiogenesis, and later by two low-Mr proteins at the anterior and posterior poles of the nucleus during sperm maturation in the epididymis. Initially, monoclonal antibodies (mAbs) PNT-1 (IgG2b) and PNT-2 (IgG2a) bound to antigens present in a cap-like configuration at the apical pole of the spermatid nucleus at step 5 of spermiogenesis. The distribution of epitopes on the nucleus expanded posteriorly until, in testicular sperm they covered the anterior pole down to the distal limits of the subacrosomal perforatorium. By contrast, sperm from the epididymis and vas deferens bound both mAbs in two distinct regions on the nucleus, one on the dorsal margin of the anterior pole, and the other in a ventral zone at the posterior pole. On SDS-PAGE and isoelectric focusing (IEF) immunoblots, both mAbs bound three major proteins with Mr of ∽80 000,77 000 and 75 000 from spermatids and testicular sperm, and proteins of Mr 50 000 and 48 000 in epididymal and vas deferens sperm. Both the high- and low-Mr protein families were recovered in germ cell nuclear/perinuclear matrices. Their mobilities on SDS-PAGE were not altered by exo- or endoglycosidases or by aminoethylation in denaturing conditions. mAb PNT-1 bound to the sperm proteins with a Ka of 3.53 ×1012 M-1 and mAb PNT-2 with a KB of 2.08 ×1012 M-1. From competition binding data, mAbs PNT-1 to -10 appeared to recognize six adjacent or overlapping epitopes on the same proteins. These data suggest the high-Mr proteins, the thecins, present at the anterior pole of haploid germ cells are processed at the onset of sperm maturation to yield two low-Mr proteins that then occupy two distinct domains at the anterior and posterior poles of the nucleus.
Spatial organization of repetitive dna sequences in the bovine sperm nucleus
Authors: Powell Don , Cran David G. , Jennings Carol , Jones Roy
Published: April 2021

During spermatogenesis, DNA in the sperm head becomes more tightly condensed as histones are replaced by protamine-like molecules. In this article, the question is asked whether, during the production of this highly differentiated cell, controls are imposed on the spatial organization of DNA within the nucleus. Heads from bull spermatozoa were isolated by a technique that removed the plasma membrane and acrosomal contents, and the DNA was induced to decondense by addition of 2-mercapto-ethanol and trypsin. Under these conditions, decondensation was induced in all regions of the head. To determine whether there was any spatial restraint on packaging of the genome, three DNA probes were used (pl.709—512, containing an interspersed repetitive sequence; pCSIH, containing a copy of the major bovine centromeric satellite sequence; pl8s and p28s, containing the 18 S and 28 S ribosomal genes) that might be expected to hybridize to different regions. Results showed that the interspersed repetitive probe hybridized to all regions of the head, whereas the ribosomal and centromeric probes hybridized to sequences that were largely confined to the equatorial region of the sperm. We conclude that organization of the genome in the bovine sperm nucleus is not random.
Interzonal microtubules are dynamic during spindle elongation
Authors: Shelden E. , Wadsworth P.
Published: April 2021

The pattern and extent of microtubule assembly during spindle elongation has been examined in PtKi cells by microinjection of biotin–tubulin and immu-nolocalization of biotin–tubulin-containing microtubules using antibodies to biotin. PtK1 cells were microinjected at 30°C, incubated for various intervals to allow incorporation of biotin–tubulin into microtubules, then lysed, fixed and stained for biotin–tubulin and total tubulin. When mid- to late anaphase cells were examined at short times postinjection, using conventional fluorescence light microscopy, rapid incorporation of biotin–tubulin was detected throughout the interzonal region, between the separating chromosomes, and in the spindle asters. Using confocal fluorescence microscopy, the segments of biotin-labeled microtubules in the interzonal region were found to be continuous with the distal, or plus-ends, of unlabeled microtubules. When telophase cells were examined, a marked decline in the extent of incorporation was apparent.

Quantitative analysis of the total length of labeled polymer in the interzonal region of cells from midanaphase through telophase further reveals that the extent of incorporation was maximal during late anaphase, and decreased during telophase. The measured rate of interzonal microtubule growth remained relatively constant during this period. Our results provide direct evidence for plus-end elongation of interzonal microtubules during spindle elongation and further reveal that interzonal microtubules are highly dynamic during late anaphase spindle elongation. The implications of these results for the mechanism of anaphase B are discussed.
Pericytes derived from the retinal microvasculature undergo calcification in vitro
Authors: Schor A. M. , Allen T. D. , Canfield A. E. , Sloan P. , Schor S. L.
Published: April 2021

Pericytes isolated from the bovine retinal microvasculature retain characteristic features of their in vivo counterparts, such as the presence of glycogen deposits, long filamentous processes, prominent microfilament bundles and the ability to display two distinct and reversible phenotypes. Time-lapse video-microscopy demonstrated that pericytes tend to overlap and aggregate, even in sparse cultures. After reaching confluence, they form multilayered areas that retract away from each other, resulting in the formation of multicellular nodules. These nodules increase in size and cellularity by going through repeated 5- to 6-h cycles of anchoring, spreading, cell proliferation and retraction. Alkaline phosphatase was not detected in pericytes at subconfluent or confluent densities, but this enzyme was expressed in areas of high cell density, such as multilayers and nodules. Pericytes synthesise and deposit an extracellular matrix at all stages of their in vitro development, including nodule formation. The matrix within the nodules contains cross-striated collagen fibres and matrix vesicles. Needle-like crystals of hydroxyapatite appear to be deposited within the matrix, thus leading to massive calcification of the nodule. Calcification, as assessed by electron microscopy, histochemical staining and X-ray microprobe analysis, occurred on plastic and collagen substrata in the absence of disodium-βkglycerophosphate. The addition of this compound at 5 or 10 mM or the use of a collagen substratum (rather than plastic), brought forward the process of nodule formation and calcification by 3–6 days. Our results suggest that retinal pericytes may differentiate in vitro along the osteogenic pathway.
Localization of actin, βspectrin, 43×103Mr and 58×103M
r proteins to receptor-enriched domains of newly formed acetylcholine receptor aggregates in isolated myotube membranes
Authors: Daniels Mathew P.
Published: April 2021

I have examined the possible involvement of specific cytoskeletal and peripheral membrane proteins in the early stages of acetylcholine receptor (AChR) aggregation in rat myotubes in culture by immunofluorescence localization of these proteins on the cytoplasmic face of isolated plasma membranes. A culture procedure utilizing selective replating of myoblasts and subsequent treatment with cytosine arabinoside was devised to obtain large, multipolar myotubes with extensive upper surfaces that are free of fibroblasts. These cultures were exposed for 4–6 h to embryonic pig brain extract (EBX) to induce AChR aggregate formation on the upper cell surface, and the AChRs were labeled with TRITC-copjugated a- bungarotoxin. Large sheets of plasma membranes from the upper cell surface were isolated by adhesion to a coverslip coated with a polypeptide adhesive (Cell-Tak) that was pressed on top of the culture. The membranes were labeled by indirect immunofluorescence with monoclonal antibodies against the 43×103Mr and 58×103Mr proteins, originally identified in the AChR-enriched membranes of Torpedo electroplaques, and with monoclonal anti bodies against isoforms of actin and βspectrin. The labeling patterns showed that all four of these proteins are concentrated in the punctate AChR-enriched domains within the aggregates, suggesting that they may be involved in the early stages of AChR aggregation. Immunofluorescence labeling with monoclonal antibodies against vinculin and clathrin, and with an antiserum to talin, showed that these proteins are also associated with AChR aggregates; however, their labeling patterns did not correspond closely to the AChR-enriched domains. Furthermore, vinculin and talin dissociated from most of the membrane during isolation. The concentration of βspectrin and actin isoforms on the cytoplasmic face of the AChR-enriched domains is consistent with the formation, early in the aggregation process, of a membrane-cytoskeleton association similar to that of erythrocytes.
Immortalization of male genital duct epithelium: an assay system for the cystic fibrosis gene
Authors: Coleman Lindsay , Harris Ann
Published: April 2021

The epithelia lining the vas deferens and epididymis are directly involved in the pathology of the autosomal recessive disease cystic fibrosis (CF). We have established culture systems for these epithelial cells. Long-term cell lines have now been generated from these primary epithelial cells by transformation with a plasmid containing an origin-defective simian virus 40 (SV40). Lines have been established from vas deferens and epididymis and both maintain expression of the CF gene.
Cytokeratins in mesenchymal cells: impact on functional concepts of the diversity of intermediate filament proteins
Authors: Markl Jürgen
Published: April 2021

Shape, motility, viscosity and organelle distribution of animal cells is regulated, or at least influenced, by fibrous proteins constituting a ‘cytoskeleton’ that involves the F-actin-based microfilaments (5–7 nm in diameter) and the microtubules (20–25 nm). In addition, most vertebrate cells contain a third type of highly organized protein bundle, the intermediate filaments (1F; 8–12 nm). Their specific role is less clear, but they certainly contribute to tensile strength of cells and, via their anchorages at desmosomes, to cell-cell interactions (for reviews, see Franke et al. 1982; Traub, 1985; Krohne and Benavente, 1986; Franke, 1987; Steinert and Roop, 1988; Nagle, 1988; Bershadsky and Vasiliev, 1988; Robson, 1989; Carmo-Fonseca and David-Ferreira, 1990). Ultrastructurally, the LFs of different cell types are very similar, but biochemical and immunological data have revealed in mammals at least seven distinct classes comprising, for example in humans, a total of approximately 40 different polypeptides: vimentin (predominantly, but not exclusively in mesenchymally derived cells), desmin (typical of most types of muscle cells), glial filament protein (GFP; predominantly in astrocytes), several different neurofilament proteins (in most neurons), peripherin (in certain neuronal cells), at least four different lamins (structural components of the nuclear lamina), and a whole panopticum of cytokeratins (present in almost all epithelial cells). A given tissue is characterized by a specific pattern of IF polypeptides, which is extensively used for cell typing in tumor diagnosis (see Kartenbeck, 1989), and as a differentiation marker in embryogenesis (e.g. see LaFlamme and Dawid, 1990).
In ultraviolet microbeam irradiations, characteristics of the monochromator and lamp affect the spectral composition of the ultraviolet light and probably the biological results
Authors: Forer Arthur
Published: April 2021

Biological conclusions recently published concerning ultraviolet (u.v.) microbeam irradiation of spindles are different from those we previously published. Several technical differences between the two sets of experiments were investigated. The spectral distributions in the light emitted from mercury-arc, xenon-mercury-arc, and xenon-arc lamps were measured, as were the spectral distributions after the light from these lamps passed through a monochromator that was set to various wavelengths and various half-band-widths. Both the source of the u.v. light and the half-band-width of the monochromator influence the spectral distribution of the light leaving the monochromator: depending on the conditions, the light leaving the monochromator is not necessarily of the same wavelength as that to which the monochromator is set. Differences in these aspects of the experiments could easily give rise to the different biological conclusions reached in the two sets of experiments.
The Golgi sorting domain of coronavirus E1 protein
Authors: Armstrong John , Patel Shaila
Published: April 2021

The coronavirus El membrane protein is confined to the Golgi after it is expressed in cells either by viral infection or via injection of synthetic RNA. We have investigated the features of the protein responsible for intracellular sorting and found that a C-terminal deletion of only 18 amino acids results in its transport to the plasma membrane. However, we have previously shown that this C-terminal region alone is not sufficient for Golgi retention. When El was fused to a cell-surface protein, Thy-1, the resulting molecule was retained in the Golgi. Various mutated forms of El whose destinations were the ER, cell surface orlysosomes were also fused to Thy-1, and in each case the fusion was sorted according to its El component alone. We argue that, in contrast to sorting signals for other membrane compartments, Golgi retention of El is not due to a single short peptide sequence. Instead, the Golgi ‘signal’ of El appears to require for its expression a domain comprising most of the sequence of the protein.
bimA encodes a member of the tetratricopeptide repeat family of proteins and is required for the completion of mitosis in Aspergillus nidulans
Authors: O’donnell Kerry L. , Osmani Aysha H. , Osmani Stephen A. , Morris N. Ronald
Published: April 2021

The recessive, temperature-sensitive bimAl mutation of Aspergillus nidulans blocks nuclei in metaphase at restrictive temperature. To determine whether the bimA product is essential, integrative transformation was used to create a mutation in the bimA gene. The mutation was maintained in a heterokaryon and the phenotype of spores produced by the heterokaryon was analyzed. Molecular disruption of the wild-type bimA gene is recessive in the heterokaryon and causes a metaphase block, demonstrating that bimA is an essential gene for mitosis. bimA was cloned by DNA-mediated complementation of its mutant phenotype at restrictive temperature, and the nucleotide sequence of a full-length cDNA was determined. A single large open reading frame was identified in the cDNA sequence, which predicts a protein containing 806 amino acid residues that is related (30.4 % identity) to the Schizosaccbaro-myces pombe nuc2+ gene product, which also is required for completion of mitosis. The sequence of the bimA gene indicates that it is a member of a family of mostly nuclear proteins that contain a degenerate 34 amino acid repeat, the TPR (tetratricopeptide repeat) gene family.
Expression in Escherichia coli of fragments of the coiled-coil rod domain of rabbit myosin: influence of different regions of the molecule on aggregation and paracrystal formation
Authors: Atkinson Simon J. , Stewart Murray
Published: April 2021

We have expressed in Escherichia coli a cDNA clone corresponding broadly to rabbit light meromyosin (LMM) together with a number of modified polypeptides and have used this material to investigate the role of different aspects of molecular structure on the solubility properties of LMM. The expressed material was characterized biochemically and structurally to ensure that it retained the coiled-coil conformation of the native molecule. Full-length recombinant LMM retained the general solubility properties of myosin and, although soluble at high ionic strength, precipitated when the ionic strength was reduced below 0.3 M. Constructs in which the ‘skip’ residues (that disrupt the coiled-coil heptad repeat) were deleted had solubility properties indistinguishable from the wild type, which indicated that the skip residues did not play a major role in determining the molecular interactions involved in assembly. Deletions from the N terminus of LMM did not alter the solubility properties of the expressed material, but deletion of 92 residues from the C terminus caused a large increase in solubility at low ionic strength, indicating that a determinant important for interaction between LMM molecules was located in this region. The failure of deletions from the molecule’s N terminus to alter its solubility radically suggested that the periodic variation of charge along the myosin rod may not be as important as proposed for determining the strength of binding between molecules and thus the solubility of myosin.
A Synopsis of the Bbitish Diatomacæ
Authors: Smith Rev. William
Published: April 2021

Amongst the organic beings whose existence the microscope has demonstrated, few possess a higher interest than the group now known as the family of Diatomaceæ. Appearing everywhere as the first-born of Life, wherever inorganic matters are fit for its development, these beings were looked upon suspiciously by early observers, and, whilst regarded by some as doubtful in nature, were looked upon by others as evidences of spontaneous generation. Their siliceous structure, which was early known, served to give them a strange resemblance to the mineral world, whilst, although endowed with motion, they presented no traces of organic tissues. The labours, however, of Ehrenberg set at rest the question of their belonging to the mineral world, and showed how great their claims were to be regarded as members of the animal kingdom. Their relationship to the Desmideæ was always apparent, and these beings were placed by Ehrenberg also in the animal world. But as evidence of the vegetable nature of the Desmideæ multiplied, so it became evident that the Diatomaceæ must follow. The fact that both these families produce sporangia as the result of conjugation in common with a large number of the confervæ, give them a common character which could hardly be assigned to beings belonging to both the animal and vegetable kingdoms. Hence Diatomaceæ are now commonly regarded as plants. But whilst zoologists have readily admitted this argument, the botanist on other grounds has rejected the Diatomaceæ from his domain. Schleiden, in his ‘ Principles of Botany,’ refuses, on the ground of the complicated and unplantlike structure of Diatomaceæ, to recognise them in the vegetable kingdom. Organisms which have thus constituted the battle-field of systematists must have more than usual interest for those who employ the instrument by which alone they can be discerned. But independent of this, there is scarcely any class of objects that present more beautiful forms, and none that invites the microscopist to a higher trial of the greatest powers of his instrument. On these accounts the present work could hardly fail to be popular from the subject on which it treats, and when to this is added the well-known reputation of the author for researches upon the Diatomaceæ, we need hardly say that the work needs no recommendation from ourselves.
On the Nature of the Torbanehill and other Varieties of, Coal
Authors: Redfern Peter
Published: April 2021

On the Nature of the Torbanehill and other Varieties of Coal. By Peter Redfern, M.D., Lond., Lecturer on Anatomy and Physiology, and on Histology, in the University of Aberdeen.

(Read at the Meeting of the British Association for the Advancement of Science at Liverpool, 1854.)
On the Reproductive Organs of certain Fungi
Authors: Currey Frederick
Published: April 2021

The existence of sexual organs in the lower orders of plants is a question which of late years has attracted much attention amongst botanists, and it is one upon which the powers of the microscope have been brought to bear with the happiest results.
On the Male reproductive Organs of Campanularia geniculata (Laomedea geniculata, Lam).
Authors: Schultze Dr. Max S.
Published: April 2021

The propagation of Campanularia geniculata, described by Lovén in 1837, * differs essentially from the mode of increase observed by V. Beneden in several species of the same genus. In the former species ciliated embryos are produced within axillary capsules, from vesicles presenting all the parts of an ovum, and after a distinct process of segmentation. These embryos, after they have quitted the tunic by which they are surrounded, and which resembles an incompletely developed polype, swim about free for some time, and precisely resemble the embryos of Medusa aurita; they then affix themselves, and grow into a polype resembling the parent animal. In the Campanularicæ, however, described by Van Beneden, medusoid creatures with tentacles, digestive and sensitive organs, are produced, also in axillary capsules; and which after quitting the capsule swim about free in the water and behave exactly like Medusæ. These were regarded by Van Beneden as the embryos. He considers that they are produced from ova, and supposes that they subsequently affix themselves, and after the obliteration and metamorphosis of some of their organs become Campanulariœ. Other observers on the contrary, particularly Nordmann and Dujardin, regard these medusiform products of the Campanularicæ as the developed forms of those polypes, believing that in the Medusœ arising in the asexual way, sexual organs are afterwards developed. The Campanulariœ consequently would have to be regarded as corresponding to the Strobila form of Medusa aurita.
An Account of the Structure and Relations of Sagitta Bipunctata
Authors: Busk G.
Published: April 2021

The minute creature to which the above name has been given, though abundant, perhaps, in all seas, and noticed so long ago as in the year 1781, has received but little attention from zoologists in general. Its curious and interesting structure, however, and doubtful position in the animal kingdom, render it a subject well worthy of further research ; and its minute size, and the extreme delicacy and transparence of its tissues, make it peculiarly an object of microscopical investigation. Though perhaps unknown, even by sight, to many of our readers, the Sagitta bipunctata will probably be met with on every part of the coast; and it may be procured, without difficulty, at any rate in fine and calm weather, by means of a small muslin towing net over the side of a boat.
On the Influence of Dilute Sulphuric Acid on the Deposit Layers of the Cell-Wall in its earliest condition
Authors: Hartig Dr. T.
Published: April 2021

In a previous paper in the same journal the author has shown that the continued multiplication of cells in the ligneous and alburnum layers, is effected by a twin pair of parent-cells belonging to each fibrous ray, the inner one of which throws off a series of sterile secondary cells towards the medulla, and the outer a similar series towards the bark.
The Micrographic Dictionary; A Guide to the Examination and Investigation of the Structure and Nature of Microscopic Objects
Authors: Griffith J. W. , Henfrey A.
Published: April 2021

We congratulate our readers upon the completion of this laborious and valuable work, whose peculiar nature renders a notice of it in our pages almost imperative. Anything like a critical review, however, of such an extensive encyclopedic volume is obviously impossible within our necessarily confined limits. Nor, perhaps, since the work has been, in part at any rate, long before the public, is such a review now demanded. But we think it incumbent upon us to point out in brief terms what we conceive to be its more striking merits and demerits, and how nearly its execution corresponds with the conception implied in its title ; and to what extent it will prove specially useful to the general microscopic observer, for whom alone we take it to be intended.