bluuebunny/crossref_metadata_2025 huggingface dataset:
Primary cell cultures from human embryonic corneas
Authors: Hyldahl Louise
Published: April 2021

This study shows that primary cell cultures can be established from corneas obtained from 8-week-old human embryos. Such corneas, even though they were obtained at an early stage of the embryonic development, are completely differentiated into three layers as in the adult cornea. Both corneal endothelial cells and stromal cells were found to attach and proliferate in vitro. Furthermore, it was possible to obtain pure uncontaminated cultures of stromal cells after five passages in vitro. It was also possible, using feeder cells as the substratum, to obtain pure endothelial cell cultures from embryonic corneas.
Secreted Glycoproteins Of Human Kidney Tumour Cells Contain Sulphated Complextype Oligosaccharides
Authors: Dennis Michael V. , Watson Cheryl B. , Heifetz Aaron
Published: April 2021

Human kidney tumour cells in culture incorporated [3H]glucosamine and 35SO4 into glycoprotein products, which were secreted into the culture medium. The effects of sodium butyrate, a known differentiation-inducing agent, on the production of these sulphated glycoproteins were studied. Cells were cultured in the absence or presence of butyrate (2 MM) in serum-containing medium, for various times, and the labelled glycoproteins were partially purified by DEAE-cellulose chromatography. Treatment of these cells with butyrate resulted in an increase in the synthesis of secreted [3H]glucosamine- and 35SO4-labelled glycoproteins over several days of culture. This same increase in levels of 35SO4 incorporation was not observed with Bie melanoma cells. Sodium dodecyl sulphate (SDS)/polyacrylamide gel electrophoresis revealed that five major glycoproteins labelled with [3H]glucosamine also were labelled with 35SO4. The major secreted glycoproteins from cells cultured in the absence or presence of butyrate over a 3-day period were similar by SDS/poly-acrylamide gel electrophoresis. Analyses of Pronase-derived glycopeptides indicated that these secreted 3H/35S-labelled glycoproteins contained sulphated oligosaccharides with terminal sialic acid → Gal → GlcNAc residues similar to glycoproteins secreted by vascular endothelial cells.
Brief extraction with detergent induces the appearance of many plasma membrane-associated microtubules in hepatocytic cells
Authors: Mesland D. A. M. , Spiele H.
Published: April 2021

In cultured H35 hepatoma cells membrane-associated cortical networks have a microtrabecular appearance as revealed by dry-cleaving. Filaments having diameters of 15 nm can be readily distinguished within these networks and have not been described previously. Microtubules are seldom observed to be part of this structure. Extraction of cells with 0.1% Saponin in microtubulestabilizing buffer produces holes in the membrane and reorganization of the networks resulting in the loss of microtrabecular structure, the loss of 15 nm filaments and the appearance of abundant membrane-associated microtubules (about 1 ·25 µm per µm2 substrate-adherent membrane). These observations were confirmed by immunolabelling experiments with affinity-purified anti-tubulin immunoglobulin G. By both fluorescence microscopy and electron microscopy it was shown that labelled tubulin in the cortical networks became organized into microtubules upon treatment with detergent. By determination of the microtubule density, expressed as µm microtubule per µm2 membrane, the effects of various conditions on microtubule occurrence were determined. The Saponin-induced appearance of microtubules in the membrane-associated network could be inhibited by: (1) 1 % and 2% glutaraldehyde, (2) O°C, (3) millimolar Ca2+, (4) absence of Mg2* (subsequent reversal of inhibition by addition of Mg24- was shown), and (5) 20µM-nocodazole (but not 20µM-colchicine). In addition to Saponin, extraction with 0·l % Nonidet P-40 or 0·l % Triton X-100 also resulted in microtubule-containing cortical networks. However, 0·1 % Triton N-101 was not effective, although holes were produced in the plasma membrane. These data provide evidence suggesting rapid polymerization of membrane-associated microtubule protein rather than detergent-induced displacement or collapse of existing microtubules. The arguments for this hypothesis and its implications are discussed.
Effect of tumour angiogenesis factors on the membrane of capillary and aortic endothelial cells: An e.s.r. spin-label study
Authors: Dodd N. J. F. , Kumar S.
Published: April 2021

Two distinct factors have been separated from an angiogenic extract of a rat Walker 256 carcinoma, one inducing proliferation and the other migration of capillary endothelial cells in vitro, but having no detectable effect on aortic endothelial cells. The influence of these factors on the order of plasma membranes of these cells was examined by electron spin resonance, using the lipophilic spin label 5-doxyl stearic acid. No detectable effect was observed on treating whole cells or isolated membranes with proliferation factor. In contrast, exposure of capillary endothelial cell membranes to migration factor caused a reduction of membrane order, particularly at temperatures above 30°C. The migration factor had no detectable effect on membrane order of aortic endothelial cells.
The three-dimensional structure of the cell wall glycoprotein of chlorogonium elongatum
Authors: Shaw P. J. , Hills G. J.
Published: April 2021

The green alga Chlorogonium elongatum, a member of the Volvocales, possesses a crystalline cell wall composed of hydroxyproline-rich glycoprotein similar to the primary cell wall glycoproteins of higher plants. Electron microscopy and computer image processing have been used to determine the crystal structure of the Chlorogonium cell wall in three dimensions to a resolution of 2·Onm. The structure is composed of heterologous dimers. Each subunit of the dimer comprises a long, thin spacer domain and a large globular domain, which is the site of the intra- and inter-dimer interactions. There are also sites of intersubunit interactions at the opposite ends of the rod domains. We suggest that the rods are composed predominantly of glycosylated polyproline helix, as has been suggested for higher plant cell wall glycoproteins and has been shown for the cell wall glycoprotein of Chlamydomonas reinhardtii, which is closely related to Chlorogonium.
A role for fibronectin in cell sorting
Authors: Armstrong Peter B. , Armstrong Margaret T.
Published: April 2021

A useful approach to the investigation of embryonic morphogenesis is the study of the factors that control cell movement in cell aggregates in organ culture. Previous studies, in which aggregates of embryonic chick heart ventricle tissue were paired in organ culture, supported the hypothesis that the associative behaviour is dominated by the mesenchymal cell (at the stages used the ventricle is composed of approximately 25% mesenchyme (Mes) and 75% myocyte tissue (My)) by virtue of this cell’s ability to establish a pericellular matrix rich in fibronectin. In aggregate pairs, the aggregate types that develop a fibronectin-rich matrix rapidly are spread over by the aggregate types that are less able to deposit fibronectin in the matrix. In sorting conditions, Mes sorts to the surface of My. This is explained as a consequence of a requirement that Mes have access to a component in the serum fraction of the culture medium for deposition of fibronectin in the matrix. It is proposed that the factor penetrates to a shallow depth in aggregates, limiting the establishment of a fibronectin-rich matrix to superficially located Mes. As fibronectin appears in the matrix, Mes becomes more cohesive than My, allowing it to exclude myocytes and establish itself as a pure tissue that increases in volume as mesenchyme cells migrating within the interior contact the surface zone, becoming immobilized and also activated to secrete fibronectin. The analysis presented includes an experimental investigation of the different elements of this hypothesis and also explores some of the predictions of the hypothesis.
Protein synthesis and its relation to the dna-division cycle in the fission yeast Schizosaccharomyces pombe
Authors: Creanor J. , Mitchison J. M.
Published: April 2021

The rate of protein synthesis has been measured with pulse labels of [3H]tryptophan in synchronous and asynchronous cultures of cdc mutants of Schizosaccharomyces pombe shifted up to the restrictive temperature. The cell cycle related fluctuations in rate that occur in normal synchronous cultures vanish when nuclear division is blocked in synchronous cultures of cdcï and cdc10. But they persist in cdcll where nuclear division continues and cleavage is stopped. We conclude that nuclear division affects the rate of synthesis and that this effect is inhibitory and probably persists for the last 40% of the cycle. When nuclear division has been blocked, the rate of synthesis continues to increase until a plateau is reached where the rate remains constant. Three size mutants of cdc2 reach the plateau at the same average protein content per cell although their initial protein contents vary over a threefold range. Comparison of these results with those from cdc 10 leads to the tentative conclusion that the plateau starts when the cells reach a critical protein/DNA ratio.
Species specificity of an enzyme determined by an erythrocyte nucleus in an interspecific hybrid cell
Authors: Cook P. R.
Published: April 2021

When a chick erythrocyte nucleus is introduced into the cytoplasm of a mutant mouse cell lacking inosinic acid pyrophosphorylase, synthesis of the enzyme is induced. The enzyme induced in this way has the characteristics of chick, not mouse, inosinic acid pyrophosphorylase.
The question of relationship between golgi vesicles and synaptic vesicles inoctopusneurons
Authors: Gray E. G.
Published: April 2021

Electron microscopy of the vertical lobe of octopus brain shows that the synaptic knobs of axons with penkarya in the median superior frontal lobe have synaptic vesicles, approximately 28 % of which are dense-cored (or granulated). In contrast, the endings of the amaenne neurons in the vertical lobe and the endings in the retina and optic lobe, both of which are derived from the retinal visual cells, have only agranular synaptic vesicles. The Golgi apparatuses of the median superior frontal penkarya have vesicles, approximately 4·3 % of which are granulated. The amaenne Golgi apparatuses have 1·5 % granulated vesicles. The visual cell Golgi apparatuses have virtually no dense-cored vesicles, only agranular ones. The question of the formation of dense-cored and agranular synaptic vesicles at the Golgi apparatus and their subsequent transport to the terminals are related to these observations.
A note on synaptic structure of the retina of Octopus vulgaris
Authors: Gray E. G.
Published: April 2021

Electron microscopy of the octopus retina shows that both types of synapse (formed by the visual cell collaterals and the efferents respectively) have synaptic membrane specializations with associated aggregations of synaptic vesicles—features usually regarded as indicative of synaptic contact. These have hitherto been considered as absent from the octopus retina. Other details of the retinal synapses are described and in addition the grouped microtubules in the initial portions of the optic axons are seen to have in association a regular pattern of micro filaments.
The fine structure of obliquely striated body wall muscles in the earthworm, Lumbricus terrestris linn
Authors: Mill P. J. , Knapp M. F.
Published: April 2021

The fine structure of obliquely striated muscle fibres from the body wall of the earthworm has been investigated. Certain details of the structure have been confirmed by cutting serial sections. The fibres contain both thick and thin myofilaments. The latter are attached to Z matenal and the 2 types of myofilament are arranged in interdigitating arrays to give rise to A- and I-bands and an H-zone similar to those in cross-striated muscle. The A-bands contain both thick and thin myofilaments and the I-bands only thin myofilaments. The Z-matenal is rod-shaped and these Z-rods, oriented perpendicular to the sarcolemma, are arranged in numerous parallel rows which run obliquely along the length of the fibre A line drawn parallel to the longitudinal fibre axis through a Z-rod in one row passes through a Z-rod in the next row. A thin, sheet-like array of myofilaments lies between 2 such Z-rods, forming a single sarcomere containing an A-band and 2 I-bands. The flat surfaces of neigh bouring sarcomeres are directly apposed to one another but, since the rows of Z-rods run diagonally along the length of the fibre, each sarcomere is displaced longitudinally with respect to the next, so that the A- and I-bands follow an oblique course, instead of a transverse course as in cross-striated muscle. Because of the regular stagger of the sarcomeres A- and I-bands are cut alternately in transverse sections. Also the sarcomeres are very narrow and are seen as bands lying perpendicular to the sarcolemma.

In the A-band a variable number of thin myofilaments (up to 12) surrounds each thick one. Cross-links have been seen between the 2 types of filaments.

In longitudinal sections an appearance similar to that seen in cross-striated muscle is obtained in one plane (perpendicular to the sarcolemma). In the plane at right angles to this (parallel to the sarcolemma) the A- and I-bands are at an acute angle to the longitudinal fibre axis. The thick myofilaments exhibit a banding of about 15 nm.

There is a system of transversely oriented tubules and peripheral vesicles with dyad-like structures occurring at the juxtaposition between the peripheral vesicles and the sarcolemma. It is concluded that this system is sarcoplasmic reticulum, and it is compared with tubular systems in other muscles.

Other cellular constituents are described, including a peripheral skeleton of fibrillar bundles.
Immunological and immuno fluorescent studies on keratin of the hair follicle
Authors: Kemp D. J. , Rogers G. E.
Published: April 2021

Keratins from the differentiating cortical cells of the guinea-pig hair follicle have been studied by immunological and immunofluorescent procedures. Antisera were prepared to the unfractionated keratin proteins from the follicle and the specificity of these antisera examined by lmmunodifTusion and lmmunoelectrophoresis. The major follicle antigens were the ‘low sulphur’ keratins, and the antibodies were specific for these and related α-keratins. Use of the precipitin reaction as a quantitative immunoassay for follicle keratins has been demonstrated. The fluorescent antibody technique has been applied to the hair follicle and the follicle antigens have been localized by this procedure in the cytoplasm of the developing cortical cells. By this technique keratin could not be detected in the proliferative cells of the bulb region of the follicle supporting the view that keratin synthesis begins only when the cells enter the upper bulb region of the follicle.
The use of antibiotics in the preparation of amphibian cell cultures from highly contaminated material
Authors: Laskey R. A.
Published: April 2021

Embryos and larvae of Xenopus laevis are contaminated with a wide range of micro-organisms from the maternal cloaca. Primary monolayer cultures prepared from them were usually destroyed by endogenous contaminants. Fungal contamination was readily eliminated by the usual procedures. The high incidence of bacterial contamination was not reduced by penicillin, streptomycin, tetracycline or combinations of the three. Contamination by Gram-negative bacilli, especially Proteus and Pseudomonas, remained. Treatment with carbenicillin, gentamicin or the combination of polymyxin E with kanamycin usually eliminated all infection. The cytotoxicity of effective antibiotics was tested.

The basic requirements of a method for eliminating endogenous bacterial contamination and the relative merits of the three effective treatments are discussed.
Electron-microscopic observations of polyoma virus-transformed mouse cells treated with specific immune serum
Authors: Negroni G. , Tilly Rita
Published: April 2021

Transformed, non-malignant cells from a polyoma virus-induced mouse fibrosarcoma were treated with immune serum raised in isologous mice, and fresh guinea-pig serum (complement). Electron microscopy showed that reduction in cell viability in vitro was associated with damage to cell membranes. The extravasated cell organelles showed only minimal changes.
The surface structure of cultured rabbit kidney cells as revealed by electron microscopy
Authors: Dimmock Elizabeth
Published: April 2021

An epithelioid line of rabbit kidney cells (RK 13), in which the distribution of blood group antigen A had previously been investigated by mixed agglutination, was chosen for studies of the structure of the cell surface. Cells were grown in monolayer culture, and thin sections were examined by electron microscopy after fixation in glutaraldehyde and osmium tetroxide and staining of the sections with lead, or uranyl and lead. Cells were also treated with ruthenium red incorporated in the osmium fixative. Other cells were fixed in lanthanum or potassium permanganate, and both stained and unstained sections were examined.

The morphology of RK 13 cell surfaces is described. There is apparently no great degree of cellular specialization. The treatment with ruthenium red resulted in dense staining of a layer of the cell surface that is not visible in conventional preparations, and sometimes in staining of the surfaces of intracellular organelles. Lanthanum permanganate fixation also revealed a dense layer on the surface of the plasma membrane; a less dense surface layer was distinguished in many cells fixed in potassium permanganate.

The reaction of ruthenium red with the cell surface is probably due to the presence of acidic glycoproteins, but the chemical specificity of the staining method is not yet clear. The nature of the material revealed by lanthanum and potassium permanaganates is also undefined. How-ever, these staining methods reveal that the cell surface is more complex than is apparent in cells prepared by conventional techniques. The additional surface layer is probably the site of many blood group substances and other compounds involved in the physiological reactions of the cell surface.
Membrane differentiation in the golgi complex of Micrasterias denticulata bréb. visualized by freeze-etching
Authors: Staehelin L. A. , Kiermayerf O.
Published: April 2021

Freeze-etched dictyosomes of the alga Micrasterias denticulata (Bréb.) exhibit a gradual increase in the density of particles on the membranes of successive cisternae from the forming to the maturing face. At the maturing face a drop in particle numbers can be observed in the peripheral regions of the cisternae, where fenestration and the packaging of the secretory products takes place.
Subsidiary components of the flagella of Chlamydomonas reinhardii
Authors: Hopkins J.M.
Published: April 2021

Some subsidiary components of flagella from the alga Chlamydomonas reinhardii have been studied in the electron microscope using frayed and partially dispersed material, negatively stained. In all, 5 distinct subsidiary structures have been observed, 3 of which are associated with the 9 pairs of outer tubules and 2 with the central pair of tubules.

Radial spokes, about 33 nm long and 5 nm in diameter, are attached at right angles to the A tubule of each outer pair and extend into the lumen of the flagellum in the direction of the central pair of tubules, but do not reach them. The spokes usually occur in pairs along the length of each A tubule. The interval between pairs is about 70 nm and that between the 2 members of each pair about 30 nm. ‘Secondary fibres’. The distal end of each spoke terminates in a hammerhead-like attachment some 10–20 nm by 5 nm with its axis parallel to the long axis of the flagellum. These hammerhead attachments are now identified with the so-called ‘secondary fibres’ previously deduced from micrographs of embedded and sectioned material. There is no evidence from the present work of a continuous secondary fibre throughout the length of the flagellum. Side arms are found attached to the A tubule of each outer pair. These arms, which occur in pairs, are roughly at right angles to the radial spokes which are also attached to the A tubules. The side arm material is distributed along the tubule at regular intervals of about 14 nm. The chemically more stable centre tubule has 2 longitudinal rows of projections, each projection being about 18 nm long with a repeat distance of about 16 nm. Occasionally, on the chemically less stable centre tubule, there is observed one row of projections which are somewhat similar to those on the other tubule.

New informationhas made it possible to reinterpret earlier work and to present a 3-dimensional picture of the external flagellum and its parts.
G and/or c-bands in plant chromosomes?
Authors: Schubert I. , Rieger R. , Döbel P.
Published: April 2021

Similarities and differences become evident from comparisons of centromeric and noncentromeric banding patterns in plant and animal chromosomes.

Similar to C and G-banding in animals (at least most of the reptiles, birds and mammals), centromeric and nucleolus-organizing region bands as well as interstitially and/or terminally located non-centromeric bands may occur in plants, depending on the kind and strength of pretreatment procedures. The last group of bands may sometimes be subdivided into broad regularly occurring ‘marker’ bands and thinner bands of more variable appearance.

Non-centromeric bands in plants often correspond to blocks of constitutive heterochromatin that are rich in simple sequence DNA and sometimes show polymorphism; they thus resemble C-bands. However, most of these bands contain late-replicating DNA. Also they are sometimes rich in A·T base-pairs, closely adjacent to each other and positionally identical to Feulgen+ and Q+ bands, thus being comparable to mammalian G-bands.

Although banding that is reverse to the non-centromeric bands after Giemsa staining is still uncertain in plants, reverse banding patterns can be obtained with Feulgen or with pairs of A·T versus G·C-specific fluorochromes.

It is therefore concluded that not all of the plant Giemàa banding patterns correspond to C-banding of mammalian chromosomes. Before the degree of homology between different Giemsa banding patterns in plants and G and/or C-bands in mammals is finally elucidated, the use of the neutral term ‘Giemsa band’, specified by position (e.g. centromeric, proximal, interstitial, terminal), is suggested to avoid confusion.
Dissociation of insect malpighian tubules into single, viable cells
Authors: Satmary William M. , Bradley Timothy J.
Published: April 2021

The Malpighian tubules of insects are generally composed of more than one cell type. In the hemipteran Rhodnius prolixus, the tubules are divided into two regions, termed the upper and lower tubules, each of which is composed of a distinct cell type. In the dipteran Aedes taeniorhynchus, primary and stellate cells are interspersed throughout the length of the tubules. We report here techniques for the dissociation of the Malpighian tubules of both of these species into single cells. Tubules are removed from the insect and placed for 1 h in insect Ringer containing elastase (Sigma, type III) at 4mg/ml. This treatment fully removes the basal lamella. Mild agitation by hand produces a suspension of single cells, which remain viable as determined by Trypan Blue exclusion. Isolated cells have been maintained in cell culture for one week. Using light and scanning electron microscopy, upper and lower tubule cells of Rhodnius and primary and stellate cells of Aedes can be distinguished on the basis of size, shape, microvillar length, and the presence or absence of intracellular crystals.
Endogenous β-galactoside-specific lectins in rabbit tissues
Authors: Harrison F. Lynne , Fitzgerald John E. , Catt James W.
Published: April 2021

Soluble β-galactoside-specific lectins have been detected in many rabbit tissues and are relatively abundant in certain organs. These lectins are apparently identical to the lectin in rabbit bone marrow, which may be involved in inter-erythroblast associations during differentiation, and are very similar in structure and activity to lectins described in a number of species both in adult and embryonic tissues. The rabbit lectin is a monomer with a relative molecular mass of approximately 12000 and has a low specific activity in the haemagglutination of trypsinized rabbit erythrocytes relative to lectins from other species, which are predominantly dimeric. In addition to the major antigen two minor isoforms of the rabbit galaptin were detected in all tissues. Since these lectins are widely distributed, it seems probable that they are involved in cellular functions that are prominent during development and also maintained in certain differentiated tissues. Experimental evidence currently available suggests that these lectins may be involved in the organization of extracellular matrix components.
Immunolocalization of tissue inhibitor of metalloproteinases (timp) in human cells characterization and use of a specific antiserum
Authors: Hembry Rosalind M. , Murphy Gillian , Reynolds John J.
Published: April 2021

A specific antiserum to pure human amniotic fluid metalloproteinase inhibitor (TIMP) was raised in a sheep. This antiserum was used to demonstrate: firstly, the immunological identity of the TIMP activities from amniotic fluid and culture medium of human foetal lung fibroblasts; and secondly, by indirect immunofluorescence, the secretion of TIMP by human foetal lung fibroblasts, chondrocytes, epithelial cells and smooth muscle cells. The phorbol ester, 12-O-tetra-decanoylphofbol-13-acetate, was used to stimulate secretion of TIMP by human foetal lung fibroblasts and the ionophore monensin was used to demonstrate intracellular accumulation of TIMP in the Golgi apparatus of these cells. These results are discussed in relation to other inhibitors of collagenase reported in the literature, which are probably identical to TIMP.
The specificity and stability of the triton-extracted cytoskeletal framework of gerbil fibroma cells
Authors: Gilbert Mark , Fulton Alice B.
Published: April 2021

Cellular meshworks and topography of gerbil fibroma cells can be preserved by gentle extraction procedures using Triton X-100. We determined the stability and specificity of these cytoskeletal frameworks by measuring extraction rate and its sensitivity to exogenous protein. Two buffers were used, which mimicked the intracellular and extracellular ionic environments. With both buffers, extraction was nearly complete at 5 min..This pattern of extraction was seen both in 5- and 9-day-old cultures. The same pattern of extraction was seen when three different dilutions of cells were examined the second day after plating. Thus, extraction rate was largely independent of minor variations in ionic composition, age in culture, or cell density. Specificity of the cytoskeletal frameworks so produced was determined by competition with two different exogenous proteins (bovine serum albumin or ovalbumin), which did not remove any additional material from the cytoskeletal frameworks, even with over 10% exogenous protein in the extraction buffer. This pattern of extraction is not unique to gerbil fibroma cells. A similar pattern of extraction was seen for a series of cells: mouse 3T3 cells, 3T6 cells and SVPY 3T3 cells. These experiments indicate that the cytoskeletal framework produced by Triton extraction under appropriate conditions is stable after extraction for a period of 10 min or longer, and that the structures are specific, in that they are not disrupted by the presence of exogenous proteins.
Selective association of an endogenous lectin with connective tissues
Authors: Catt J. W. , Harrison F. Lynne
Published: April 2021

Using indirect immunofluorescence we have localized an endogenous P-galactoside-specific lectin in resin-embedded rabbit tissue sections. The pattern of lectin distribution correlates well with biochemical estimations of lectin levels, being abundant in intestine, lung and heart tissue and relatively less abundant in skeletal muscle, liver and kidney. In all tissues lectin is found in connective tissue associated with fibroblasts and the extracellular matrix, and at the periphery of morphologically recognizable smooth muscle cells. The lectin is abundant in skin, intestine and blood vessels, where connective tissue forms the tissue architecture. It is also abundant in heart, where it is particularly associated with the capillaries and lung, where it is also found in alveolar cells. Discrete localization of lectin occurs in areas of connective tissue where epithelial elements are differentiating, such as the crypts of Lieberkuhns in the small intestine and hair follicles in the skin. From these observations we suggest that in cells of mesenchymal origin these endogenous lectins may play a role in the elaboration or organization of the extracellular matrix that regulates tissue differentiation in a number of embryonic and adult tissues.
A novel procedure for pattern analysis of features present on freeze-fractured plasma membranes
Authors: Appleyard S. T. , Witkowski J. A. , Ripley B. D. , Shotton D. M. , Dubowitz V.
Published: April 2021

We have used statistical methods for the analysis of two-dimensional point patterns to derive quantitative descriptions of the distributions of caveolae on freeze-fractured muscle fibre membranes. One method was based on a quadrat analysis while the second was a new procedure that we have called the interpoint distance analysis. We show that the latter analysis can unambiguously distinguish random, clustered and dispersed patterns and that a single parameter can be derived that can be used to compare different distributions. It is readily applicable to patterns containing several hundred points. Practical details of the method are given and a simple algorithm that can be implemented on a microcomputer is provided. The interpoint distance analysis should prove generally useful in situations where the two-dimensional distribution of objects has to be quantified.
Responses of cultured cardiac myocytes to lysosomotropic compounds and methylated amino acids
Authors: Decker R. S. , Decker M. L. , Thomas V. , Fuseler J. W.
Published: April 2021

Cardiac myocytes whose lysosomes had been pre-labelled with acridine orange were exposed to either L-amino acid methyl esters (L-leucine or methionine methyl ester) or to ‘lysosomotropic’ weak bases (chloroquine, methylamine, and NH4CI) for 1 h. Both types of interventions dilated lysosomes equally and inhibited proteolysis to varying degrees. The weak bases produced no apparent alterations in the acridine orange staining, whereas the methylated amino acids induced a marked redistribution of the fluorescent dye from lysosomes into the myoplasm, suggesting that they may have provoked a change in lysosomal membrane permeability. A brief exposure to weak bases failed to enhance acid proteinase secretion into the culture medium but apparently inactivated cellular cathepsin B activity. In contrast, methylated amino acids induced no alterations in acid proteinase activity or the cellular distribution of the two proteolytic enzymes. Lastly, weak bases markedly elevated intralysosomal pH as measured with fluorescein dextran, while only modest rises were observed after amino acid methyl ester treatment. The present observations imply that amino acid methyl esters represent a new class of reagents with actions distinctly different from those of chloroquine and NH4CI, and they may provide a unique and valuable means of studying secondary lysosomal function in cell culture.
Simultaneous ultrastructural localization of ag-nor (nucleolar organizer region) proteins and ribonucleoproteins during mitosis, in human breast cancerous tissues
Authors: Ploton D. , Menager M. , Adnet J. J.
Published: April 2021

The simultaneous ultrastructural localization of the Ag-NOR proteins and ribonucleoproteins (RNP) was carried out during mitosis in human breast cancerous tissues using a one-step silver staining method followed by acetylation.

Owing to the good preservation of the cells and to the high precision of the Ag staining we were able to follow the behaviour of the argyrophilic nucleolar components from prophase to late telophase.

During all the phases of mitosis the Ag-NOR proteins were associated with fibrillar RNP component (without visible fibrillar centre), which originated, during prophase, from the dense fibrillar component of the nucleolus and then integrated within the periphery of some metaphasic chromosomes as irregular structures. The Ag staining of these structures was homogeneous but lower compared to those of nucleoli during prophase.

The RNP sheath that covered the chromosomes was never silver stained.

From anaphase to early telophase the argyrophilic RNP structures disposed within the periphery of the chromatin mass formed roundish and then spherical structures in which the Ag-NOR proteins were clustered as centrally localized spheres.

These findings showed the usefulness of our method for the precise study of the spatial relationship between the Ag-NOR proteins and the chromosomes. Thus we were able to show striking segregation of both the argyrophilic structures at the periphery of the chromatin mass and of the Ag-NOR proteins within these structures. This suggests that a high degree of the structural organization of the future nucleus is already present during anaphase. This could be related to the rapid resumption of ribosomal RNA synthesis.
Effect of microtubule-destroying drugs on the spreading and shape of cultured epithelial cells
Authors: Domnina L. V. , Rovensky J. A. , Vasiliev J. M. , Gelfand I. M.
Published: April 2021

The role of microtubules in the spreading of cells from the liver-derived IAR2 rat cell line was studied. Cells in control medium seeded on a flat isotropic glass surface rapidly spread to form discoid shapes. Spreading in colcemid-containing medium was disorganized and delayed; partial reversal of spreading was observed. Nevertheless, even in the presence of colcemid the cells finally spread to discoid flattened shapes. IAR2 cells in medium without colcemid spread not to discoid but to elongated shapes under three different sets of conditions: (1) when the cells were forced to spread on narrow strips of adhesive glass surface between two non-adhesive lipid films; (2) when the cells spread on the poorly adhesive surface of poly(HEMA)-covered glass; (3) when the cells spread on the usual glass surfaces in medium containing cytochalasin D. Addition of colcemid to the media reversed the polarized spreading under the first two conditions; colcemid did not reverse the formation of the elongated cell shape acquired by the cells spreading in cytochalasin-containing medium. Effects of microtubule-destroying drugs on the spreading of epithelial and fibroblast cells are compared and discussed. It is suggested that microtubules are essential for the stabilization of the spread state of those attached cytoplasmic processes and lamellae that do not have numerous and stable cell-substratum contacts, e.g. the processes formed at the early stages of spreading or the elongated processes of polarized cells. Possibly, microtubules stabilize the non-contracted state of the actin cytoskeleton in these processes.
Microtubules and root protophloem ontogeny in wheat
Authors: Eleftheriou E. P.
Published: April 2021

Protophloem ontogeny in roots of Triticum aestivum has been investigated ultrastructurally. Each protophloem pole consists of three cells, a protophloem sieve element and two companion cells, all originating from a single precursor cell usually having a pentahedral shape. This protophloem mother cell (PMC) undergoes two successive asymmetrical divisions: the first one gives rise to a smaller cell that will differentiate into a companion cell, and a larger one that divides again asymmetrically yielding another companion cell and a protophloem sieve element. The latter divides once more, but now symmetrically, increasing the number of cells.

Both asymmetrical and symmetrical divisions are preceded by preprophase microtubule bands (PMBs), well demarcated by a great number (more than 100 profiles in a single band section) of microtubules (MTs). The plane of a PMB coincides with that of the succeeding cell plate, which fuses with parent walls at sites previously occupied by the PMB. The strict correspondence between PMB and cell plate suggests that at cytokinesis the latter bisects the PMB cortical zone. The possible role of PMB cortical zone in positioning the cell plate and guiding its expanding edges towards predetermined sites is discussed in relation to recent discoveries in other anatomical situations. The plane of PMBs (and hence of divisions) changes from one division to the next, so that the three successive divisions occur in three spatial planes transversely to each other. This change is probably influenced by cell polarity.

Prior to each asymmetrical division peri-nuclear MTs were observed besides the MTs of the PMB. They appear before the PMB organization and persist throughout preprophase, but they change their position and orientation in response to the transition from PMB to the spindle organization.
Interlocked bivalents in reconstructed metaphase i cells of bread wheat
Authors: Heslop-Harrison J. S. , Bennett M.D.
Published: April 2021

Complete reconstructions of all the bivalents were made from electron micrographs of serial sections through six pollen mother cells at metaphase I of meiosis in Triticum aestivum (hexapioid bread wheat). At least two of these metaphases contained interlocked pairs of bivalents. In one, two ring bivalents were interlocked, while in another a rod bivalent ran through the centre of a ring bivalent. Two other groups of bivalents were too closely appressed to allow separation into individual bivalents and may have contained interlocks. Meiosis in other anthers of the same plants examined by light microscopy was considered normal. The frequency of interlocking found was much higher than reported from light-microscope spreads.

Not all interlocks in metaphase I cells need adversely affect meiosis, but knowledge of their regularity and form may facilitate understanding the processes of chromosome pairing.
The aggregation of trypsinized BHK21 cells
Authors: Edwards J. G. , Campbell Janice A.
Published: April 2021

The aggregation in suspension of freshly trypsinized cultured cells (BHK21) has been studied by microscopic and electronic counting techniques. Comparison of cells grown in suspension with cells grown on glass suggests that the aggregation may not be an artifact of tryptic action, but may reflect an adhesive property of the cell surface which survives the dispersal procedure.

The aggregation is very strongly inhibited at low temperature, and resembles in this respect the re-aggregation of cells from trypsinized embryonic tissues. There is no detectable delay in aggregation at 37 °C, and cells pre-incubated at 37 °C do not aggregate at 2 °C. These results are inconsistent with the view that the temperature-dependence of cell aggregation in this system is for resynthesis of surface components.

The aggregates are readily redispersed by proteolytic enzymes. The adhesions in the aggregates thus resemble in this respect the adhesions between cells in many tissues.

Trypsin and chymotrypsin are of similar effectiveness in dispersing the aggregates, but di-isopropylphosphoryl trypsin is completely ineffective, indicating that the enzyme activity of the proteins is involved in dispersal.
A method of obtaining a suspension of intact parenchymal cells from adult rat liver
Authors: Gallai-Hatchard Jennifer J. , Gray G. M.
Published: April 2021

The perfusion of liver with either citrate or tetraphenyl boron to remove Ca2+ or K+ or with a solutionof high osmolarity and alkaline pH yields plenty of cells but they are all damaged.Perfusion of the liver with hyaluronidase and collagenase followed by incubation of liver slices in the same enzyme solution produced a high yield of cells (25 %, w/w, of liver) of which only about 1 % were undamaged. However, perfusion with 0·3 % hyaluronidase, 0·3 % collagenase and 0·1 % trypsin in phosphate-buffered saline (excluding Mg2+ and Ca2+ followed by incuba-tion at 25 °C of the chopped liver gave a small yield (2–4%, w/w) of undamaged cells which were not permeable to eosin for up to an hour when suspended in culture medium containing 2 % bovine serum albumin.
The fine-structural organization of the brush border of intestinal epithelial cells
Authors: Mukherjee T. M. , Staehelin L. A.
Published: April 2021

The fine structure of the brush border of intestinal epithelial cells of the mouse has been studied with both normal sectioning and freeze-etching techniques.

Freeze-etching reveals the plasma membrane of the microvilli as consisting of a continuous layer, that is split during the cleaving process, in which numerous particles, 5–9 nm in diameter, are embedded, while other particle-like structures, with diameters of 7–10 nm, appear attached to the true outer membrane surface. The mucopolysaccharide surface coats of the microvilli show up more clearly in sectioned material than in freeze-etched specimens.

Inside each microvillus 2 different filament systems can be demonstrated: (1) bundles of fairly closely packed and straight core microfilaments, which lead into the tip of the microvillus, and (2) short cross-filaments. Under suitable conditions the core microfilaments display a sub-unit structure with a repeating distance of approximately 6 nm. The diameter of a microfilament can vary along its length from 6 to 11 nm. Two strands of globular particles wound helically around each other seem to make up each microfilament. These and other data support the idea that the core microfilaments are actin-like. No substructure has been found on the cross-filaments, which have an orientation approximately radial to the axis of the microvilli and seem to be attached at one end to the core microfilaments and at the other to the inner surface of the microvillous membrane.

The interwoven terminal web filaments also show no substructure. They form a continuous flexible platform-like structure into which the bundles of core microfilaments extend. Some terminal web filaments appear attached to the plasma membrane between the microvilli. It is suggested that the core microfilaments represent mechanical supporting elements and that the terminal web and cross-filaments are tensile elements of the brush border. In addition all 3 filament systems may also be involved in possible contractile movements of the microvilli.
The anchoring of nuclei by cytoplasmic microtubules inAcetabularia
Authors: Woodcock C. L. F.
Published: April 2021

The giant unicellular alga Acetabularia enters the reproductive phase of growth when the cap has completed its expansion. The primary nucleus then divides and the resulting secondary nuclei take up fixed equidistant positions in the coenocytic cap cytoplasm. These positions are visible as colourless areas in the otherwise green caps.

Studies on the cytology and ultrastructure of the caps at this stage showed that each secondary nucleus was surrounded by a layer of cytoplasm which was free from chloroplasts and mitochondria, but which contained many microtubules, some of which were closely associated with the nuclear envelope, while others appeared to touch the plasmalemma adjacent to the cell wall. (Microtubules are not a regular feature of Acetabularia cells; they have not been reported in the stems, young caps or cysts, and vegetative growth is not inhibited by colchicine.)

The function of the microtubules was investigated by treatment with colchicine or vinblastine sulphate, both of which depolymerize microtubules in many other systems. Administration of these drugs at 10−3 M and greater concentrations had the following effects: (1) the colourless areas were lost as chloroplasts and mitochondria invaded the cytoplasm around each secondary nucleus; (2) the nuclei began to migrate from their fixed positions; and (3) cyst formation (in which the cytoplasm cleaves into uniformly sized cysts, each containing a single secondary nucleus) either was inhibited, or proceeded abnormally.

It is therefore proposed that a major function of these transitory microtubules is to anchor the secondary nuclei at fixed equidistant positions in the cytoplasm. This function is probably mediated by their association both with the nuclear envelope and cell membrane.
The density of ribosomes bearing messenger rna in phage-infected and normal bacteria
Authors: Fenwick M. L.
Published: April 2021

Newly made viral RNA in lysates of Escherichia coli infected with phage R17 was studied as a model messenger RNA. The viral RNA that was associated with groups of 1, 2, 3 and 4 ribosomes banded in caesium chloride at densities of approximately 1·68, 1·65, 1·645 and 1·635 g cm-3 respectively and the single ribosomes of uninfected cells at 1·62. The density of infected ribosomes was restored to 1·62 by treatment with RNase. These values are consistent with the presence of a single molecule of viral RNA in each group.

In uninfected bacteria the mean density of the smaller polysomes exceeded that of actino-mycintreated or RNase-treated ribosomes by about 0·005 g cm−3. Similarly, pulse-labelled RNA associated with single ribosomes or pairs banded at a density only marginally higher than that of the bulk of the single ribosomes. If any RNA was attached to the ribosomes it probably amounted to not more than one fifth of the size of the phage RNA for each ribosome pair.
The analysis of malignancy by cell fusion: II. hybrids between ehrlich cells and normal diploid cells
Authors: Bregula U. , Klein G. , Harris H.
Published: April 2021

When Ehrlich ascites cells were fused with diploid fibroblasts, isolated directly from the animal, the resulting hybrid cells regularly produced progressive tumours. However, an analysis of a range of clonal populations of these hybrid cells, each derived from a separate primary fusion, revealed that the chromosomal constitution of these cells was highly unstable; all cell populations were found to have already undergone substantial chromosome losses by the time enough cells were available to permit chromosomal analysis. Thus, although these hybrid cells were highly tumorigenic, the tumours arising from them were not composed of cells with complete parental chromosome sets, but of cells from which some chromosomes had been eliminated.
The intramembrane structure of septate junctions based on direct freezing
Authors: Kachar Bechara , Christakis Nicholas A. , Reese Thomas S. , Lane Nancy J.
Published: April 2021

Smooth septate junctions from the midgut of the cricket, Acheta, and the horseshoe crab, Limulus, as well as Hydra-type septate junctions from the epidermis of Hydra have been studied by freeze-fracture after direct freezing using the liquid helium-cooled copper block/slam freezing method. The exoplasmic fracture face at both types of septate junction exhibits rows of closely packed but irregularly shaped intramembrane particles. Complementary to these particle rows, on the protoplasmic fracture face, are sharply defined grooves with a periodic variation in depth and width that was conspicuous in Hydra but less well defined in arthropods. The closely packed, irregular particles on the exoplasmic faces could represent plastically deformed portions of transmembrane proteins pulled through the bilayer during freeze-fracture. On the basis of this interpretation, the grooves on the protoplasmic faces represent a confluence of the bilayer disruptions occurring during fracturing. The structures observed here are different from those reported in replicas of glutaraldehyde-fixed and glycerol-cryoprotected tissue, in which the intramembrane junctional components partition with the protoplasmic face and often assume the appearance of continuous cylinders. This comparison illustrates some of the artifacts associated with freezefracturing and shadowing. On the basis of a comparison of freeze-fracture replicas and sections of lanthanum-infiltrated tissues, the relationship between intramembrane junctional components and intercellular septal elements is analysed.