bluuebunny/crossref_metadata_2025 huggingface dataset:
Electron- and light-microscope observations on the spleen of the newt Triturus cristatus: the surface topography of the mitotic chromosomes
Authors: Davies H. G. , Tooze J.
Published: April 2021

In polychromatophil erythroblasts, and in cells containing little or no haemoglobin, the surfaces of the mitotic chromosomes frequently bear finger-like projections averaging about 0·1 μ in diameter. Both the mitotic and interphase chromosomes in polychromatophil erythro-blasts from some newts bear, in addition, extensive sheets of chromatin, about 315-465 A thick, depending on fixation, bounded on each side by nuclear envelope-like fragments. The remarkably constant width of the sheets in all cells indicates that chromosomes contain a constructional unit of fixed dimensions. This unit may have general significance, since other workers have found envelope-limited sheets of similar dimensions in one plant and several animal species. An hypothesis concerning the construction of chromosomes is discussed. It seeks to relate the thickness of the sheets of chromatin to the average diameter of the majority of the finger-like processes.

The large intranuclear granules, 400 Å in diameter, in polychromatophil erythroblasts survive in polysome-like configurations in dividing cells.
Observations on the substructure of flagellar fibres
Authors: Grimstone A. V. , Klug A.
Published: April 2021

The substructure of the outer and central fibres of the flagella of Trichonympha and other flagellates has been examined by electron microscopy, using negatively-stained material. Periodicities revealed in the micrographs have been analysed by studying the optical diffraction patterns obtained from them. Outer and central fibres appear to be essentially similar in sub-structure and are made up of a number of longitudinal filaments which form the wall of the hollow fibres. The filaments are 40–50 Å apart, centre to centre, and are beaded, the beads (or subunits) giving rise to an axial repeat along the filaments of 40 Å and a basic surface lattice of 40 × 50 Å. In collapsed or disintegrating fibres no other substructural features are usually visible. In intact fibres, however, additional longitudinal periodicities are apparent. An 80-Å spacing is prominent, and there is commonly also a strong 160-Å repeat. These appear to be orders of a fundamental 480-Å period. The long spacings are thought to arise as departures in the arrangement of the subunits from the simple 40 × 50 Å lattice, the perturbations probably occurring both radially and in the plane of the surface of the fibre. In the central fibres additional material is present, spaced at 160 Å and probably corresponding to the central sheath previously described from sectioned material. Observations on the arms of the outer fibres are described.
Ciliary organelles and associated fibre systems in Euplotes eurystomus (Ciliata, Hypotrichida): I. Fine structure
Authors: Gliddon R.
Published: April 2021

Euplotes eurystomus bears 8 rows of single, non-motile simple cilia, 18 ventral cirri, and a row of adoral membranelies. Each cirrus contains 40–120 cilia in tight hexagonal packing, and each membranelle consists of 3 rows of 10–40 cilia; the component cilia adhere together in life, but no means of adhesion, other than small protuberances of the ciliary membrane, has been seen.

The kinetosomes of compound organelles, but not of the simple cilia, are associated with rootlet fibres. The kinetosomes of the component cilia of compound organelles are linked together at two levels by characteristic arrays of fibres. Single rootlet microtubules (about 20 mμ in diameter) interconnect individual kinetosomes of adjacent membranelles, but the rootlet microtubules arising from cirri are found as bundles, each of which corresponds to one of the fibres seen in the light microscope. The bundles of microtubules of most cirri arise at dense bars near the edge of the cirrus base, and terminate at or near the pellicle, without interconnecting cirri directly or indirectly. The rootlet microtubules of the anal cirri run the length of the base of the cirrus near the inner end of the kinetosomes and join into large bundles; these run anteriorly to join with each other and attach to the distal end of the membranelle row. The anal cirri and the adoral zone of membranelles form one structurally united part of the locomotor apparatus.
A spiral sleeve in rotifer cilia tips
Authors: Dickson M. R.
Published: April 2021

The fine structure of cilia in the gut of the rotifer, Philodina roseola, has been studied. The cilia display the usual structures for the first 9 μ. of their length, but for the last 3 μ the shaft consists of plasma membrane clothing an unusual spiral sleeve. The sleeve is made up of 5 parallel fibres twisted in a left-handed helix about the long axis of the cilium.
Development and determination of hairs and bristles in the milkweed bug, oncopeltus fasciatus (lygaeidae, hemiptera)
Authors: Lawrence P. A.
Published: April 2021

The term ‘ organule ‘ is proposed as an English equivalent for the German word ‘ Kleinorgan The different organules on the third sternite of Oncopeltus are described: larvae possess innervated bristles and special sensilla termed ‘chemosensilla’, whereas the adult develops, in addition, a dense population of non-innervated hairs. The hairs, bristles and ‘chemosensilla’ each develop from mother cells which undergo a particular series of differentiative divisions. The course of events is described for each type of organule.

Electron-microscopic studies of the fine structure of the outgrowing hairs are described. As in the outgrowth of a scale, longitudinal arrays of microtubules and bundles of fibres are found in the first cytoplasmic process of the trichogen cell. Experiments show that bristle determination occurs between the onset of the moult and the proliferative cell divisions in the epidermis. The period of hair determination is found to occur later than that of the bristles and to be later than the proliferative cell divisions in the last larval stage.

A discussion of the results includes a review of the knowledge of differentiative divisions concerned in the formation of organules in other groups of insects, and a consideration of Wigglesworth’s theory of bristle determination.
Chloroplast ribosome deficient mutants in the green alga Chlamydomonas reinhardiand the question of chloroplast ribosome function
Authors: Boynton J. E. , Gillham N. W. , Chabot J. F.
Published: April 2021

The 2 chloroplast ribosome deficient mutants of Chlamydomonas reinhardi, ac-20and cr-I form low levels of 66-s and 70-s chloroplast ribosome monomers compared to wild type, but differ in that cr-I also accumulates substantial amounts of the large (54-s) chloroplast subunit. Unlike wild type, ac-20, cr-I and the double mutant ac-2ocr-i make little or none of the CO2-fixing enzyme ribulose diphosphate carboxylase (RuDPCase) when grown in the light with acetate and CO2 as carbon sources (mixotrophic growth) whereas all 3 mutant genotypes form at least some enzyme when grown photosynthetically with CO2 as the sole carbon source (phototrophic growth). All 3 mutant genotypes also show a characteristic defect in the organization of their chloroplast lamellar system when grown mixotrophically, but their chloroplast membrane organization approaches that of wild type when grown phototrophically. No change in the level of chloroplast ribosomes accompanies these changes in chloroplast organization and RuDPCase formation. We propose that the pleiotropic effects on chloroplast structure and function in mixotrophically grown cells of the mutants result because their chloroplast ribosomes synthesize certain specific chloroplast components with low efficiency, whereas they perform this function with greater efficiency when the cells are grown phototrophically.
Variation in the nuclear sodium concentration of newt oocytes during maturation
Authors: Muir C. , Whitley J. E.
Published: April 2021

Measurements of the sodium content of single oocyte nuclei of Triturus cristatus carnifex at various stages of maturation, show a rise in concentration in eggs of the size 0·5–0·6 mm diameter. When the nuclei are isolated rapidly under paraffin, the absolute values obtained for the concentration are much lower than have been previously reported for this species.
The concentrations of dry matter in mitotic apparatuses in vivo and after isolation from sea-urchin zygotes
Authors: Forer A. , Goldman R. D.
Published: April 2021

We have measured the concentrations of dry matter in mitotic apparatuses (MA) in vivo and after isolation from the same cell type. The isolation medium was hexylene glycol plus buffer. The MA were from sea-urchin zygotes (Echinus esculentus Linn, and Psammechinus miliaris Gmelin), and measurements were made using interference microscopy.

MA as isolated have much lower concentrations of dry matter than do MA in vivo. The dry mass concentrations of isolated MA vary with the pH of the isolation medium, ranging from about 20 % of the in vivo concentration (at pH 7·3) to about 60 % of the in vivo concentration (atpH5·3).

The isolated MA were further characterized. Evidence is presented which suggests that non-specific cytoplasmic material adsorbs to MA, and thus that at least some of the material in isolated MA is not derived from in vivo MA. Some MA components are apparently changed during the isolation procedure: MA lysed in low pH (high mass) medium and quickly transferred to high pH (low mass) medium have higher concentrations of dry matter than do MA lysed in high pH medium.

The isolation media as generally used do not have enough buffering capacity: the pH changes after the isolation.

These data suggest that the isolation procedures need be improved before studies of isolated MA can give data relevant to the chemistry of in vivo MA. We discuss the problem of obtaining functional isolated MA, and also the relevance of our data to previous work on MA from other species.
Intracellular mosaicism (nuclear-/mitochondrial+) for thymidine kinase in mouse l cells
Authors: Clayton D. A. , Teplitz R. L.
Published: April 2021

An electron-microscope and ultracentrifuge examination of mitochondria and mitochondrial DNA from Cl-I-d cells has demonstrated that mitochondria possess the enzyme(s) necessary for incorporation of thymidine and its analogues. These cells lack the nuclear thymidine kinase and are routinely grown in high levels of 5-bromodeoxyuridine (Budr) to select against wildtype revenants. When these cells are grown in the presence of 5-bromodeoxyuridine the mitochondrial DNA incorporates the thymidine analogue into both strands of the DNA duplex while the nuclear DNA shows no incorporation. Approximately 13 % of the total thymidines of the M-DNA are replaced by 5-bromodeoxyuridine and the DNA exhibits the form expected for normal mouse mitochondrial DNA. If the cells are grown in normal medium the mitochondrial DNA regains its normal base composition. Comparison of mitochondrial morphology under these 2 conditions reveals that the presence of 5-bromodeoxyuridine results in a distortion of the cristae similar to the effect induced by chloramphenicol. It is hypothesized that the incorporated mutagen (5-bromodeoxyuridine) may be altering the protein products of mitochondrial DNA. The results suggest (1) the existence of a separate and distinct thymidine kinase in the mitochondria of mouse L cells and (2) mutagenic but reversible effects of Budr incorporated into mitochondrial DNA.
Premature chromosome condensation: a mechanism for the elimination of chromosomes in virus-fused cells
Authors: Rao P. N. , Johnson R. T.
Published: April 2021

The fate of prematurely condensed chromosomes (PCC) that are induced following fusion between a mitotic and an interphase cell was studied in the homokaryons of HeLa and 2 glycine-requiring mutants of Chinese hamster ovary cells. The data indicate: (1) the prematurely condensed chromosomes are often incorporated into daughter nuclei of the fused cells and hence they are functionally retained by the progeny; (2) in general, the only viable products of fusion between mitotic and interphase cells are those where PCC has been induced; (3) fusion between mitotic and S-phase cells leads to the most rapid loss of chromosomes, while the homophasic fusions tend to produce more stable hybrids with regard to their chromosome number.
The effect of cell population density on nutrient uptake and cell metabolism: a comparative study of human diploid and heteroploid cell lines
Authors: Griffiths J. B.
Published: April 2021

The possibility that contact inhibition of growth in cultures of human diploid cells is influenced by the effects of cell crowding on nutrient uptake by the cells was investigated. Two human lung cell lines were compared, the diploid line MRC-5 and the heteroploid line L-132. In pre-confluent cultures the ability of these 2 cell types to accumulate amino acids was very similar. Post-confluent L-132 cells showed very little change from the pre-confluent cultures but the ability of MRC-5 cells in post-confluent cultures was greatly reduced. The intracellular concentrations of various amino acids necessary to achieve the maximum rate of protein synthesis were found. These values were identical for sparse and crowded cultures but due to the reduced uptake ability of crowded MRC-5 cells a far higher external amino acid concentration was required in post-confluent cultures. This meant that although amino acids did not become growth-limiting until over 80 % utilized in pre-confluent cultures, in post-confluent cultures they became growth-limiting when only 50 % utilized.

Although protein synthesis was significantly affected by extracellular amino acid concentration and cell crowding, thus contributing towards the effect of contact inhibition of growth, DNA synthesis was shown to be the major metabolic function in contact inhibition. Increased cell density had a very inhibitory effect on DNA synthesis in MRC-5 cultures, but not in L-132 cultures, and this was unaffected by extracellular amino acid and glucose concentration.
The effect of injections of lysophosphatidyl choline into white matter of the adult mouse spinal cord
Authors: Hall Susan M.
Published: April 2021

The action of lysophosphatidyl choline, LPC, on myelinated fibres in the dorsal white matter of the spinal cord of the adult mouse has been studied electron microscopically, and compared with the recently described activity of LPC in the peripheral nerve fibre.

Control injections of sterile isotonic saline and injections of LPC both produced oedematous zones in the white matter; within these zones, many fibres exhibited the characteristic changes of Wallerian degeneration. After injection of LPC, however, an area of demyelination was observed, extending within and beyond the Wallerian degeneration. Ultrastructurally, demyelination involved progressive disruption of the previously-compact sheath, observed initially as a splitting of the intraperiod line within 30 min. Subsequent breakdown was-indicated by the appearance of strands of 4-6 nm repeat lamellar material, itself further degraded through quintuple- and triple-layered lamellar units to disorganized membranous networks around undamaged axons.

The significance of the demyelinating activity of LPC is discussed in terms of its known action in in vitro systems of isolated central nervous tissue, and its action in vivo in the peripheral nervous system.
Localization of freeze-fracture planes of yeast membranes
Authors: Hereward F. V. , Northcote D. H.
Published: April 2021

In freeze-etch replicas the position of the plane of fracture along membranes is controversial; in an attempt to clarify the situation we have examined drops of yeast suspension frozen at − 160 °C, fractured and then freeze-substituted with a solution of osmium tetroxide in acetone. The drops were embedded in Araldite and sections were cut perpendicular to the plane of fracture to show where the cleavage had occurred.

At the tonoplast the membrane split and the plane of fracture ran between the 2 darkly staining lines. At the plasmalemma the cleavage plane was not clearly localized. The inner half of the membrane remained with the protoplast and darkly staining material could be seen on the wall. However, in some places the dark line at the surface of the protoplast could be resolved into 2 lines. The cleavage plane appeared to change at the invaginations of the plasmalemma, where on fracture the whole membrane sometimes remained attached to the wall.
Movement, fine structure, and fusion of pseudopods of an enclosed amoeba, Difflugiella sp
Authors: Griffin J. L.
Published: April 2021

In Difflugiella sp., strain F-20, a small amoeba enclosed in a flexible mantle, pseudopods extended through a mouth or aperture and seemed to function only for movement and feeding. Pseudopods from different cells fused on contact and cell clumps shared common pseudopods and moved in a co-ordinated way. During locomotion, pseudopods or pseudopod complexes usually exhibited an activity cycle of 3 phases: anterior extension with the tip firmly adhering; stable hold as other pseudopods advanced; and flaccid posterior retraction. While distal adhesive tips advanced, proximal unattached parts of pseudopods simultaneously shortened as the cell body advanced.

Microtubules were numerous in pseudopods within the mouth but extended for only 1–2 μm into pseudopods up to 20–30 μm long. Microfilaments were present where pseudopods adhered to the substratum, to the mantle, or to bacteria and were also associated with pinocytotic invaginations. Pseudopod ground plasm was either reticulate or amorphous; no axial rods or aligned filaments related to pseudopod rigidity were seen.

Simultaneous pseudopod adhesion, extension, and proximal shortening apparently account for locomotion or cell body translation of Difflugiella. While some similarities to other amoeboid systems were noted, the need for detailed studies on different types of organisms or cells is emphasized.
The dogfish neuromuscular junction: dual innervation of vertebrate striated muscle fibres?
Authors: Bone Q.
Published: April 2021

In the myotomal muscles of the dogfish, Scyliorhinus canicula, there are 2 major types of fibre. The red fibres at the periphery of the myotome receive a distributed en grappe pattern of innervation. There are subjunctional folds at these endings, and the nerve terminals contain vesicles around 50 nm in diameter. In contrast to this, the white twitch fibres of the myotome are innervated focally, by 2 nerve fibres passing to the same motor end-plate. These 2 fibres contain vesicles of different types. One type of nerve terminal contains vesicles around 50 nm in diameter; these terminals resemble those upon the red fibres. The other contains vesicles up to too nm in diameter, frequently possessing a dense core. It is suggested that the white twitch fibres of dogfish are innervated by 2 separate axons, possibly containing different transmitter substances.
Effects of denervation on the ultrastructure of insect muscle
Authors: Rees D. , Usherwood P. N. R.
Published: April 2021

The structure of normal and denervated muscle fibres in the metathoracic retractor unguis muscle of the locust (Schistocerca gregaria) has been examined. Section of the 2 motor neurons which innervate this muscle results initially in muscle hypertrophy but this is followed about 4 days post neurotomy by progressive atrophy. Atrophy of the retractor unguis muscle is characterized by a decrease in muscle volume and degeneration of muscle organelles, e.g. mitochondria, sarcoplasmic reticulum, transverse tubular system, protein filaments, etc. During its later stages the degeneration of the denervated muscle is possibly assisted by the phagocytic action of haemocytes which invade the muscle.
Nematode-induced syncytium—a multinucleate transfer cell
Authors: Jones M. G. K. , Northcote D. H.
Published: April 2021

The formation and structure of a syncytium induced by the potato cyst-nematode (Heterodera rostochiensis Woll.) in potato roots is described.

At the permanent feeding site of the nematode larva, usually in the root cortex, the larva pierces a cell with its mouth stylet and injects saliva. Cell wall dissolution occurs to incorporate neighbouring cells into a syncytium. A column of cells is incorporated towards the vascular tissue. Centripetal advance is limited by the lignified xylem, then syncytial spread continues laterally along xylem parenchyma and pericycle cells.

Wall protuberances form on syncytial walls adjacent to conducting elements. This indicates the syncytium is a multinucleate transfer cell, and by ingesting syncytial contents the larva is the nutrient sink. As syncytial expansion occurs, sieve elements are crushed and probably cease to function, hence protuberance development continues only against xylem elements.

Cell alterations on incorporation into the syncytium involve expansion, loss of cell vacuole, nuclear hypertrophy and a proliferation of cytoplasmic organelles free to move through wall gaps into the communal cytoplasm.

‘Boundary formations’ and microtubules are associated with the growing ends of protuberances, and appear to be involved in their synthesis. Fibrillar material, possibly cellulose microfibrils, occurs between the plasmalemma and the membrane of the ‘boundary formation’, and the forming protuberance.

To induce the formation of the syncytium, the larva controls the differentiation of unspecialized cells to cells with a specific physiological function. The occurrence of wall protuberances suggests that transfer cells form as a response to solute flow.
A light-microscope study of the action of cytochalasin b on the cells and isolated cytoplasm of the characeae
Authors: Williamson R. E.
Published: April 2021

The reversible inhibition of cytoplasmic streaming in cells of Nitella translucens by cytochalasin B was found to be accompanied by an increase in the number of mini-vacuoles in the endoplasm. Fibrils thought to drive the streaming were still present in inhibited cells. In droplets of cytoplasm obtained from cut cells of Chara corralina, the final number of fibrils formed was not sensitive to cytochalasin B, although the motility of these fibrils was highly so. The movement of organelles in the absence of visible fibrils and the rotation of chloroplasts were also inhibited. The evidence for the involvement of the microfilament system in cytoplasmic streaming is discussed. The way in which such microfilaments seem to be operating in characean cells differs from the most likely mechanism for their operation in other cytochalasinsensitive processes.
The effect of actinomycin d in vivo upon peripheral nucleoli and other nuclear organelles in oocytes of Triturus cristatus
Authors: Snow M. H. L.
Published: April 2021

Exposure of the ovaries of Triturus cristatus to actinomycin D at a concentration of 100 /tg/ml causes characteristic changes in the peripheral nucleoli and other nuclear organelles in oocytes of o·6–1·1 mm diameter. Viewed with the light microscope untreated oocytes contain nucleoli that stain uniformly with a variety of dyes. They also appear homogeneous under phasecontrast optics. After 2 or 4 h of in vivo incubation with actinomycin D, oocyte sections stained with Haidenhain’s haematoxylin or viewed under phase-contrast optics show nucleoli composed of 2 regions. The more heavily stained or contrasted zone is crescent-shaped and directed away from the nuclear membrane. Neither sections stained with azure B bromide nor gallocyanin chrome alum show this feature. Ribonuclease digestion does not eliminate or alter it. Autoradiography with [3H]uridine indicates that all recently synthesized RNA is lost from the nucleolus during actinomycin D treatment. The zonation is not therefore a reflexion of RNA distribution. During recovery from actinomycin D poisoning there is a reduction in the degree of zonation shown by nucleoli which re-establish a normal appearance some 48 h after treatment.

Electron microscopy of peripheral nucleoli in oocytes sampled during this treatment indicates that the zonation is not associated with reorganization of ultrastructural components. During incubation with actinomycin D the coarse granules (20 nm diameter) are completely lost from the nucleolus. There is associated shrinkage of the nucleolus which after treatment is found to consist entirely of fibrils (5 nm thick) and small granules. The reappearance of the coarse granules during recovery is completed in about 48 h. It is thought that the loss of the granular component during treatment represents the movement of the 30-s precursor and the 18-s ribosomal unit from the nucleolus.

Some 20–30 μm inside the nucleus of untreated oocytes is a region containing many spheroidal bodies, less than 1·0 μm diameter. They have been termed micronucleoli and consist of granules 2·5–5 nm in diameter and fibrils of similar thickness. Actinomycin D treatment causes these components to segregate and eventually (within 24 h of treatment) the granular component is extruded. This component reappears during the second day after treatment. It is postulated that these micronucleoli represent the sites at which the 30-s ribosomal precursor undergoes its final maturation. The segregation of components induced by actinomycin D is probably the morphological manifestation of an abnormal metamorphosis of this precursor.

Treatment with actinomycin D also induces the immediate formation within the nucleus of crystalline bodies composed of lamellae 16 nm wide, 4 nm thick and with a centre-to-centre spacing of 8–10 nm. They are not present 24 h after treatment. They are thought to represent a protein fraction normally associated with periods of intense RNA synthesis.
Cytoplasmic localization during storage and translation of the mrnas of transition protein 1 and protamine 1, two translationally regulated transcripts of the mammalian testis
Authors: Morales Carlos R. , Kwon Yunhee K. , Hecht Norman B.
Published: April 2021

During spermatogenesis in mammals, the transcripts of transition protein 1 (TP 1) and protamine 1 (Prm 1) are under translational regulation. Following their transcription in round spermatids, the mRNAs for TP 1 and Prm 1 are stored in the cytoplasm from 3–7 days before being translated towards the end of spermatogenesis. To test the hypothesis that the inactivation or activation of transcripts during spermiogenesis could be mediated by mRNA compartmentalization in the cytoplasm of spermatids, light and electron microscopy were used to localize, by in situ hybridization, the cellular and subcellular sites of stored and translated mRNAs for these two testisspecific transcripts. During early spermiogenesis (before step 7) nuclear transcripts of both TP 1 andPrm 1 were seen. After step 7 the TP 1 and Prm 1 mRNAs were only detected in the cytoplasm. Throughout spermiogenesis the cytoplasmic mRNAs were not localized to any membrane-bound organelles such as the endoplasmic reticulum or mitochondria or to non-membrane-bound structures such as the chromatoid body. These studies demonstrate that the translational arrest of the TP 1 and Prm 1 mRNAs is not primarily controlled by compartmentalized storage in the cytoplasm of spermatids. Moreover, when translation of these mRNAs occurs in elongated spermatids, the mRNAs are present throughout the cytoplasm.
High molecular weight tau: Preferential localization in the peripheral nervous system
Authors: Georgieff Irene S. , Liem Ronald K. H. , Mellado Wilfredo , Nunez Jacques , Shelanski Michael L.
Published: April 2021

Using epitope mapping we have demonstrated that a high molecular weight protein (Mr ∼115× 103) present in brain and spinal cord is a member of the tau family of microtubule-associated proteins. Antibodies directed against the amino-terminal, middle and carboxyl-terminal portions of tau recognize this protein. A limited survey of neuronal tissues has shown that this high molecular weight tau protein is present in brain, spinal cord, dorsal root ganglia, dorsal and ventral roots and peripheral nerves. High molecular weight tau protein is expressed at higher levels in spinal cord than in brain and is the only form of tau detected in the adult peripheral nervous system.
Centrosome and microtubule dynamics during meiotic progression in the mouse oocyte
Authors: Messinger Susan M. , Albertini David F.
Published: April 2021

The disposition, function and fate of centrosomes were analysed in mouse oocytes undergoing in vitro meiotic maturation, using multiple-label fluorescence microscopy. Oocytes fixed at various points during meiotic progression were double labeled with either human centrosome-specific antibody, 5051, and anti-tubulin antibodies or 5051 and MPM-2 antibodies in order to evaluate the microtubule nucleation capacity and phosphorylation status of centrosomes during this process. Double labeling with anti-tubulin antibodies revealed two populations of centrosomes that undergo stage-specific changes in number, location and microtubule nucleation capacity in relation to spindle assembly and cytoplasmic events. Specifically, one population was consistently associated with chromatin throughout meiotic maturation whereas a second population of cytoplasmic centrosomes exhibited maximal numbers and nucleation capacity at prometaphase and anaphase of meiosis-I. Quantitative evaluation of cytoplasmic centrosomes indicated increased numbers during the transition from diakinesis to prometaphase and metaphase to anaphase and total disappearance during telophase. Colocalization studies with MPM-2 revealed that centrosomes were always phosphorylated. However, at metaphase of meiosis I and II the microtubule nucleation capacity of centrosomes was diminished. These results suggest the existence of two discrete populations of centrosomes in the mouse oocyte that are coordinately regulated to subserve aspects of microtubule organization relative to both nuclear and cytoplasmic events.
The inner dynein arm complex: Compatible images from freeze-etch and thin section methods of microscopy
Authors: Burgess S. A. , Carter D. A. , Dover S. D. , Woolley D. M.
Published: April 2021

A study has been made of the inner dynein arm complex in the sperm flagellum of Gallus domesticus. It has been found that images of the complex made from highly contrasted, approx. 20 nm, sections are very largely compatible with images made from replicas of rapidly cryofixed material. This suggests that neither technique seriously distorts the native state. From both types of image, we conclude that the most proximal group of inner dynein arm heads (IDA 1) is related to spoke SI and consists of 3 heads with fine connections to the B-tubule. IDA 2 consists of 2 such heads and is related to spoke S2. IDA 3 is apparently single headed and lies close to the A-attachment of spoke S3. This arrangement of ID As repeats every 96 nm. Between the IDAs and outer dynein arms (ODAs), lying close to IDAs 2 and 3, is a strap-like linkage to the next B-tubule; it is argued that this represents the circumferential link, nexin. In sections, but not consistently in replicas, IDA 2 lies closer to the plane of the nexin link than does IDA 1. In the presence of ATP and vanadate, little change is seen in the IDA complex except for an ill-defined alteration to IDA 1. It is speculated that the apparently smaller size of the inner arm heads (compared with the ODA major subunit) may have functional implications in relation to maximal sliding velocity.
Combined effects of extracellular matrix and growth factors on NBT-II rat bladder carcinoma cell dispersion
Authors: Tucker Gordon C. , Boyer Brigitte , Valles Ana Maria , Thiery Jean Paul
Published: April 2021

Using the rat bladder carcinoma cell line NBT-II we showed that collagens but not laminin and fibronectin were able to induce cell scattering. Acidic fibroblast growth factor and transforming growth factor alpha also promoted NBT-II cell dispersion on glass or tissue culture plastic. We have now further analysed the scatter response to these two growth factors in the presence of extracellular matrix molecules. In the presence of growth factors, no peripheral single-cell dispersion occurred on fibronectin and laminin, although time-lapse video analyses revealed intense cell mingling and motility inside the monolayer forming around NBT-II aggregates. Patterns of strings or files of cells protruding from the monolayer were often observed. The presence of a scattering activity in the complex acellular extracellular matrix deposited by NBT-II cells themselves strongly suggested that substratum conditioning was responsible for this effect. On the other hand, the two growth factors accelerated collagen-mediated NBT-II individual cell dispersion and locomotion in a reversible way. As a marker of cell dissociation, we studied desmosome distribution in aggregate cultures: desmosomes were present in aggregates formed in suspension even in the presence of growth factors, whereas internalization occurred after cell-to-substratum contact. On laminin or fibronectin and in the presence of growth factors, peripheral cells inside the halo of NBT-II aggregates did not exhibit desmosome linkages. These observations suggest that scatter effects per se are dependent on the composition of the extracellular matrix. In particular, on a substratum nonpermissive for direct cell translocation, individual cell dispersion can be replaced by en bloc patterns of migration following substratum conditioning by the cells.
Enhanced expression of fibronectin by cmd/cmd chondrocytes and its modulation by exogenously added proteoglycan
Authors: Tsukahara Takashi , Okamura Miya , Suzuki Sakaru , Iwata Hisashi , Miura Takayuki , Kimata Koji
Published: April 2021

Cartilage matrix deficiency (cmd/cmd) in mice is a genetic disorder associated with the failure of chondrocytes to synthesize the core protein of cartilage proteoglycan monomer (cartilage-PG). Immunohistochemical and biochemical analyses revealed enhanced accumulation of fibronectin in culture of cmd/cmd chondrocytes as well as in mutant cartilage tissue in vivo. Purified cartilage-PG, when added exogenously to a culture of cmd/cmd chondrocytes, caused a reduction in abnormal accumulation of fibronectin over several subsequent days of culturing. Using a fibronectin cDNA probe, we showed that, on the basis of total RNA, the fibronectin mRNA level was four or eight times higher in cmd/cmd chondrocytes than in the normal. The level of fibronectin mRNA in cmd/cmd cells was lowered by culturing the cells in the presence of added cartilage-PG. These findings suggest that the abnormal accumulation of fibronectin in the mutant cell culture is primarily due to elevation of fibronectin mRNA level, and that cartilage-PG in the extracellular matrix may affect the regulation of fibronectin biosynthesis at the steady-state level of mRNA.
Evidence for kinesin-related proteins in the mitotic apparatus using peptide antibodies
Authors: Sawin Kenneth E. , Mitchison Timothy J. , Wordeman Linda G.
Published: April 2021

To identify kinesin-related proteins that may be important for mitotic function in embryonic and tissue culture cells we have generated polyclonal antibodies to two synthetic peptides corresponding to conserved regions of the kinesin motor domain. In Xenopus eggs we have identified a family of microtubule-binding proteins, recognized by one or both affinity-purified peptide antibodies but not by monoclonal antibodies that recognize conventional kinesin heavy chain. Like kinesin, most of these proteins bind to microtubules only upon addition of AMP-PNP or nucleotide depletion and are released upon subsequent addition of ATP. At least one protein, however, exhibits markedly distinct properties, binding readily to microtubules in the absence of AMP-PNP and/or nucleotide depletion. We also report that, unlike antibodies to conventional kinesin, the peptide antibodies to the kinesin motor domain immu-nofluorescently label spindles and kinetochores in mitotic tissue culture cells, suggesting that kinesin-like proteins may have important roles in chromosome movement and mitosis.
Down-regulation of scatter factor in MRC 5 fibroblasts by epithelial-derived cells: A model for scatter factor modulation
Authors: Kamalati Tahereh , Thirunavukarasu Birunthi , Wallace Andrew , Holder Nigel , Brooks Robert , Nakamura Toshikazu , Stoker Michael , Gherardi Ermanno , Buluwela Lakjaya
Published: April 2021

Scatter factor/hepatocyte growth factor (SF/HGF) is a multifunctional cytokine produced by embryonic fibroblasts and other mesenchymal cells that affects the growth and/or the movement of certain epithelia. Here we report that expression of scatter factor activity by MRC 5 cells, a strain of normal human embryonic lung fibroblasts, is greatly reduced as a result of co-culture of these cells with SVK14, an SV40-transformed human kératinocyte cell line. Using a cDNA probe to the βchain of human HGF, we have found that the fall in SF activity in MRC 5/SVK14 co-cultures is accompanied by the loss of SF/HGF transcripts. As the inhibition of SF activity coincides with the disappearance of SF/HGF transcript, we conclude that inhibition of the SF activity expressed by MRC 5 cells by co-culture with SVK14 involves transcriptional regulation.
Transport of the cationic fluorochrome rhodamine 123 in an insect’s Malpighian tubule: indications of a reabsorptive function of the secondary cell type
Authors: Meulemans W. , Loof A. De
Published: April 2021

The pathway of rhodamine 123 was examined after injection into Sarcophaga flies and after in vitro labeling of the Malpighian tubules. After in vitro labeling the primary cells only retained this potential-sensitive dye for a short period while all secondary cells accumulated the dye from the tubule lumen. In vivo the secondary cells also accumulated rhodamine 123 from the lumen, but the primary cells in the distal parts of all four tubules retained the dye for prolonged periods. This was most pronounced in the distal part of the anterior Malpighian tubules, where rhodamine 123 was eventually precipitated on the luminal concretions. Rhodamine 123 initially accumulated in the secondary cell mitochondria and eventually in intensely fluorescing vesicles, probably lysosomes. No evidence for endocytotic processes from the lumen was found using Lucifer Yellow CH, fluorescent dextrans and fluorescent albumin. Prior incubation with the ionophores valinomycin, nigericin, CCCP (all 1 μg/ml), dinitrophenol (1 mM) and NaN3 (10−2 M) inhibited the selective accumulation of rhodamine 123 to a large extent while monensin (1-5 μg/ml) showed little inhibitory effect. Furthermore, only cationic and no anionic or neutral dyes were accumulated by the secondary cells. In the fleshfly Calliphora and the fruitfly Drosophila, the dye rhodamine 123 also selectively accumulated in the secondary cells, as well in vitro as in vivo.
Tracking of cell surface receptors by fluorescence digital imaging microscopy using a charge-coupled device camera: Low-density lipoprotein and influenza virus receptor mobility at 4°C
Authors: Anderson Catherine M. , Georgiou George N. , Morrison Ian E. G. , Stevenson Gregory V. W. , Cherry Richard J.
Published: April 2021

A fluorescence imaging system, based on using a cooled slow-scan CCD camera, has been developed for tracking receptors on the surfaces of living cells. The technique is applicable to receptors for particles such as lipoproteins and viruses that can be labeled with a few tens of fluorophores. The positions of single particles in each image are determined to within 25 nm by fitting the fluorescence distribution to a two-dimensional Gaussian function. This procedure also provides an accurate measure of intensity, which is used as a tag for automated tracking of particles from frame to frame. The method is applied to an investigation of the mobility of receptors for LDL and influenza virus particles on human dermal fibroblasts at 4°C. In contrast to previous studies by FRAP (fluorescence recovery after photo bleaching), it is found that receptors have a low but measurable mobility at 4°C. Analysis of individual particle tracks indicates that whilst some receptors undergo random diffusion, others undergo directed motion (flow) or diffusion restricted to a domain. A procedure is proposed for subdividing receptors according to their different types of motion and hence determining their motional parameters. The finding that receptors are not completely immobilised at 4°C is significant for studies of receptor distributions performed at this temperature.
Visualization of DNA within mitochondria by osmium-ammine staining of mouse duodenal crypt cells
Authors: Liu Dong Feng , El-Alfy Mohamed , Leblond Charles Philippe
Published: April 2021

Previous investigators have examined mitochondrial DNA (mtDNA) in the electron microscope (EM) after extraction from mitochondria and rotary shadowing. We have observed mtDNA in situ by the osmiumammine procedure for specific staining of DNA in the EM. The procedure was modified to improve the regularity of the staining and then applied to the rapidly dividing cells present in mouse duodenal crypts. In the stained sections of these cells, 25% of the mitochondria exhibited discrete reactive filaments. The filaments, whether observed directly or in stereopairs, appeared either irregular or arranged into distinct patterns, some of which were similar to those previously described after rotary shadowing of duplicating mtDNA: namely, simple and double circular figures, displacement loops and supercoiled forms. The filaments could be traced in serial sections of the same mitochondria and, therefore, were not artifacts. Moreover, their disappearance after DNase digestion demonstrated that they were composed of DNA. It is concluded that mtDNA can be visualized by the modified osmium-ammine technique and may show patterns that can be interpreted as phases in its replication.
A monoclonal antibody, raised against mammalian centrosomes and screened by recognition of plant microtubule organizing centers, identifies a pericentriolar component in different cell types
Authors: Chevrier Véronique , Komesli Sylviane , Schmit Anne-Catherine , Vantard Marylin , Lambert Anne-Marie , Job Didier
Published: April 2021

We have used monoclonal antibodies raised against isolated native calf thymus centrosomes to probe the structure and composition of the pericentriolar material. To distinguish prospective antibodies as specific to conserved elements of this material, we screened clones by their identification of microtubule organizing centers (MTOCs) in different animal and plant cells.

Among the clonal antibodies that reacted with MTOCs in both plant and mammalian cells, we describe one (mAb 6C6) that was found to immunostain centrosomes in a variety of bovine and human cells. In cycling cells this signal persisted through the entire cell cycle. Microscopy showed that the mAb 6C6 antigen was a component of the pericentriolar material and this was confirmed by biochemical analysis of centrosomes. Using immunoblot analysis of protein fractions derived from purified components of centrosomes, we have characterized the mAb 6C6 antigen as a 180 kDa polypeptide. We conclude that we have identified a protein component permanently associated with the pericentriolar material.

Surprisingly, monoclonal antibody 6C6 also stained other mitotic organelles in mammalian cells, in a cellcycle-dependent manner. During prometaphase and metaphase the antibody stained both centrosomes and kinetochores. At the onset of anaphase the kinetochore-specific staining dissociated from chromosomes and was subsequently redistributed onto a newly characterized organelle, the telophase disc while the centrosomal stain remained intact. It is not known if the 180 kDa centrosomal protein itself redistributes during mitosis, or if the pattern observed represents other antigens with shared epitopes.

The pericentriolar material is thought to be composed of conserved elements, which appeared very early during the evolution of eukaryotes. Our results strongly suggest that mAb 6C6 identifies one of these elements.
Development of a probenecid-sensitive Lucifer Yellow transport system in vacuolating oat aleurone protoplasts
Authors: Wright K. M. , Davies T. G. E. , Steele S. H. , Leigh R. A. , Oparka K. J.
Published: April 2021

Oat aleurone protoplasts, maintained in liquid culture over a period of five days, have been shown to develop an uptake system capable of transporting the membrane-impermeant probe Lucifer Yellow CH (LYCH). The dye was completely excluded from the densely cytoplasmic, early developmental stages but its uptake increased exponentially after the protoplasts had been cultured for between 2 and 5 days. Culturing induced vacuolation and uptake of the dye was largely restricted to highly vacuolate protoplasts. No cytoplasmic staining was evident. In vacuolate protoplasts uptake was linear with time and saturated with increasing substrate concentration. Low temperature, and addition of the drug probenecid to the incubation medium, completely eliminated LYCH uptake. In contrast to unconjugated LYCH, LYCH-dextrans (Mr10,000 and 40,000) were excluded from the protoplasts.

The data negate simple diffusion and fluid-phase endocytosis as possible candidates for dye uptake and suggest, instead, the development of highly co-ordinated membrane transport systems on both plasmalemma and tonoplast.
Morphometric studies of pancreatic acinar granule formation in NCTR-Balb/c mice
Authors: Weintraub H. , Abramovici A. , Amichai D. , Eldar T. , Ben-Dor L. , Pentchev P. G. , Hammel I.
Published: April 2021

NCTR-Balb/c mice are afflicted with a cholesterol lysosomal storage disorder stemming from a defect in intracellular cholesterol processing. The clinical and biochemical abnormalities expressed in the mice resemble Niemann-Pick type C and D disorders in humans. One of the proposed mechanisms to explain the pathophysiology of the disorder implies a defect in the process of membrane transport that normally takes place in the vesicular movement of cholesterol to specific target sites in the cell.

Secretory granule formation in pancreatic acinar cells is one of the biological processes known to involve massive membrane flow. Thus, we have undertaken a morphometric study of the regranulation mechanism in the pancreatic acinar cells of the mutant mice, as a way of studying cellular membrane movement.

Electron micrographs of pancreatic acinar cells from mutant and normal mice were taken at several time points after extensive degranulation induced by pilocarpine injection. Two hours after stimulation the pancreatic cells demonstrated a complete loss of granules, and at later time points newly formed granules appeared. Identical unit granule volumes were observed in both groups, indicating that the progranules were of normal size. However, the rate of granule formation and maturation was reduced in the mutant mice, which might be the result of a defect in membrane function.
Dynamics of transmembrane proteins during Sindbis virus budding
Authors: Pavan Antonio , Covelli Eugenio , Pascale Maria C. , Lucania Giuseppe , Bonatti Stefano , Silva Pedro Pinto Da , Torrisi Maria Rosaria
Published: April 2021

Label-fracture and immunogold fracture-flip techniques are used to address at the ultrastructural level the dynamics of viral and cellular transmembrane proteins during the budding of Sindbis virus on the plasma membrane of infected cells. Immunolabeling with anti-Sindbis spike antibodies shows that the viral proteins are mostly in clusters, all associated with budding viruses. Ultrastructural observation of the unlabeled freeze-fractured plasma membranes shows that membrane particles aggregate over the budding viruses. These results indicate that the concentration of viral transmembrane proteins gives rise to a parallel concentration of membrane particles. Immunolabeling with anti-CD8 antibodies of cells expressing by transfection the CD8 transmembrane protein and infected with Sindbis virus shows absence of labeling on the particle aggregates over the forming virions. These findings indicate the exclusion of CD8 proteins from the portions of the membrane where budding occurs.