bluuebunny/crossref_metadata_2025 huggingface dataset:
Paracrine interactions of chondrocytes and macrophages in cartilage degradation: articular chondrocytes provide factors that activate macrophage-derived pro-gelatinase B (pro-MMP-9)
Authors: Dreier Rita , Wallace Shona , Fuchs Susanne , Bruckner Peter , Grässel Susanne
Published: April 2021

Cells of the monocyte/macrophage lineage are involved in the development of inflammatory joint diseases such as rheumatoid arthritis. This disease is characterized by cartilage degradation and synovial membrane inflammation with a progressive loss of joint function. The pathological processes are still not well understood. Therefore it would be interesting to develop a suitable experimental in vitro model system for defined studies of monocyte/macrophage and chondrocyte interactions at the molecular level. For that purpose we cocultured chondrocytes from adult human articular cartilage with human monocytes and macrophages for defined periods of time in agarose without addition of serum. We performed zymographic and western blot analysis of culture medium, completed by quantitative RT-PCR of each chondrocyte, monocyte and macrophage RNA, respectively. The reliability of the newly established coculture systems is confirmed by causing a clear decrease of intact aggrecan in the coculture medium plus concurrent appearance of additional smaller fragments and a reduction of chondrocyte aggrecan and collagen II gene expression in the presence of monocytes. In culture medium from cocultures we detected active forms of the matrix metalloproteinases MMP-1, MMP-3 and MMP-9 accompanied by induction of gene expression of MMP-1, membrane type 1 MMP (MT1-MMP) and tissue inhibitor of metalloproteinase 2 (TIMP-2) in chondrocytes. No gene expression of MMP-9 was detectable in chondrocytes, the enzyme was solely expressed in monocytes and macrophages and was downregulated in the presence of chondrocytes. Our results suggest that MMP-9 protein in coculture medium originated from monocytes and macrophages but activation required chondrocyte-derived factors. Because addition of plasmin, a partial activator of pro-MMP-3 and pro-MMP-1, enhanced the activation of pro-MMP-9 and pro-MMP-1 in cocultures but not in monocultured macrophages, and the presence of MMP-3 inhibitor II prevented pro-MMP-9 activation, we assumed a stepwise activation process of pro-MMP-9 that is dependent on the presence of at least MMP-3 and possibly also MMP-1.
The abundant retinal protein of the Chlamydomonas eye is not the photoreceptor for phototaxis and photophobic responses
Authors: Fuhrmann Markus , Stahlberg Alke , Govorunova Elena , Rank Simone , Hegemann Peter
Published: April 2021

The chlamyopsin gene (cop) encodes the most abundant eyespot protein in the unicellular green alga Chlamydomonas reinhardtii. This opsin-related protein (COP) binds retinal and was thought to be the photoreceptor controlling photomovement responses via a set of photoreceptor currents. Unfortunately, opsin-deficient mutants are not available and targeted disruption of non-selectable nuclear genes is not yet possible in any green alga. Here we show that intron-containing gene fragments directly linked to their intron-less antisense counterpart provide efficient post-transcriptional gene silencing (PTGS) in C. reinhardtii, thus allowing an efficient reduction of a specific gene product in a green alga. In opsin-deprived transformants, flash-induced photoreceptor currents (PC) are left unchanged. Moreover, photophobic responses as studied by motion analysis and phototaxis tested in a light-scattering assay were indistinguishable from the responses of untransformed wild-type cells. We conclude that phototaxis and photophobic responses in C. reinhardtii are triggered by an as yet unidentified rhodopsin species.
Rapid dynamics of the microtubule binding of ensconsin in vivo
Authors: Bulinski J. Chloë , Odde David J. , Howell Bonnie J. , Salmon Ted D. , Waterman-Storer Clare M.
Published: April 2021

Microtubule-associated proteins (MAPs) are proteins that reversibly bind to and regulate microtubule dynamics and functions in vivo. We examined the dynamics of binding of a MAP called ensconsin (E-MAP-115) to microtubules in vivo. We used 5×GFP-EMTB, a construct in which the microtubule-binding domain of ensconsin (EMTB) is fused to five copies of green fluorescent protein (GFP), as a reporter molecule amenable to the use of fluorescent speckle microscopy. Fluorescent speckle microscopy (FSM) sequences and kymograph analyses showed rapid dynamics of speckles comprised of 5×GFP-EMTB in untreated cells. By contrast, in detergent-lysed cytoskeletons, speckles were not dynamic. Since detergent-lysed cytoskeletons differ from living cells in that they lack both ATP and dynamic microtubules, we used azide treatment to substantially reduce the level of ATP in living cells and we used Taxol to halt microtubule dynamics. Both treatments slowed the dynamics of 5×GFP-EMTB speckles observed by FSM. We also used fluorescence recovery after photobleaching (FRAP) to quantify the half-time of binding and dissociation of the 5×GFP-EMTB chimera and to compare this half-time to that of the full-length MAP molecule. In untreated cells, the tg of either 5×GFP-EMTB or full-length GFP-ensconsin was similarly rapid (∼4 seconds), while in ATP-reduced and Taxol-treated cells, tg was increased to 210 seconds and 40 seconds, respectively. In detergent-extracted cells no recovery was seen. Consistent with the rapid dynamics of 5×GFP-EMTB measured with fluorescent speckle microscopy and FRAP, we estimated that the affinity of the MAP for microtubules is ∼40 μM in untreated living cells, compared with ∼1 μM in vitro. However, KD,app was not significantly changed in the presence of azide and was increased to 110 μM in the presence of Taxol. To test whether changes in the phosphorylation state of cellular proteins might be responsible for altering the dynamics of ensconsin binding, we used FSM to monitor staurosporine-treated cells. Staurosporine treatment substantially halted dynamics of 5×GFP-EMTB speckles along MTs. Our results show that ensconsin is highly dynamic in its association with microtubules, and its microtubule association can be altered by in vivo phosphorylation events.
Proteasome-mediated regulation of the hDlg tumour suppressor protein
Authors: Mantovani Fiamma , Massimi Paola , Banks Lawrence
Published: April 2021

The Dlg tumour suppressor protein is intimately involved in the control of cell contact and polarity. Previous studies have shown that hDlg is a target for a number of viral transforming proteins. In particular, the high risk human papillomavirus (HPV) E6 proteins target hDlg for proteasome-mediated degradation, an activity that appears to contribute to HPV-induced malignancy. However, little information is available concerning the normal regulation of hDlg. In this study we have investigated the role of the proteasome in the regulation of endogenous hDlg protein levels in epithelial cell lines. We demonstrate that hDlg is, indeed, degraded via the proteasome both in the presence and absence of HPV, in a fashion that is dependent on the ability of the cells to form cell junctions. By western blot and immunofluorescence analysis we show that hDlg is efficiently degraded in isolated cells; however, upon cell-cell contact, hDlg is both hyper-phosphorylated and stabilised. Strikingly, in both transformed rodent cells and undifferentiated cervical cancer cells, this ability to stabilise Dlg upon increased cell density is lost. These results demonstrate a complex pattern of hDlg regulation by phosphorylation and proteasome degradation in response to cell contact. Loss of this regulation probably represents a significant step in the development of malignancy.
snRNP protein expression enhances the formation of Cajal bodies containing p80-coilin and SMN
Authors: Sleeman Judith E. , Ajuh Paul , Lamond Angus I.
Published: April 2021

Splicing snRNPs (small nuclear ribonucleoproteins) are essential sub-units of the spliceosome. Here we report the establishment of stable cell lines expressing fluorescently tagged SmB, a core snRNP protein. Analysis of these stable cell lines has allowed us to characterize the nuclear pathway that leads to snRNP accumulation in nuclear speckles and has identified a limiting nucleolar step in the pathway that can be saturated by overexpression of Sm proteins. After nuclear import, newly assembled snRNPs accumulate first in a subset of Cajal bodies that contain both p80-coilin and the survival of motor neurons protein (SMN) and not in bodies that contain p80-coilin but lack SMN. Treatment of cells with leptomycin B (LMB) inhibits both the accumulation of snRNPs in nuclear bodies and their subsequent accumulation in speckles. The formation of Cajal bodies is enhanced by Sm protein expression and the assembly of new snRNPs. Formation of heterokaryons between HeLa cell lines expressing Sm proteins and primary cells that usually lack Cajal bodies results in the detection of Cajal bodies in primary cell nuclei. Transient over-expression of exogenous SmB alone is sufficient to induce correspondingly transient Cajal body formation in primary cells. These data indicate that the level of snRNP protein expression and snRNP assembly, rather than the expression levels of p80-coilin or SMN, may be a key trigger for Cajal body formation.
The role of soft connective tissue in the response of pig articular cartilage in organ culture to excess of retinol
Authors: Barratt M. E. J.
Published: April 2021

The action of excess rerinol on articular cartilage from growing pigs was studied in organ culture. Retinol had little or no effect on explants of articular cartilage alone, but if the explants were cut so as to include some of the marrow tissue in the invasion cavities, or were cultivated near or in conlact with capsular tissue, retinol caused extensive degradation of the cartilage matrix, as indicated by loss of metachromatic staining properties. Many chondrocytes were released from their capsules and assumed a fibroblast-like form. Two types of regeneration were seen. In control explants that included part of the invasion zone, cells below the explant laid down a metachromatic matrix; in similar explants cultured in the presence of retinol, a non-metachromatic osteoid-like tissue was formed at this site. There was little recovery when retinol-treated explants were transferred to normal medium, although both osteoid and chondroid tissue were sometimes regenerated.
Ultrastructural and cytochemical manifestations of protein synthesis in the peripheral sarcoplasm of denervated and newborn skeletal muscle fibres
Authors: Gauthier Geraldine F. , Schaeffer Susan F.
Published: April 2021

In muscle fibres of the rat diaphragm, there is widespread accumulation of free ribosomes and rough-surfaced endoplasmic reticulum in the subsarcolemmal sarcoplasm at 28 days following section of the phrenic nerve. A corresponding accumulation of basophilic material is visible at the periphery of the fibres when examined with the light microscope. Both the ribosomes and the basophilic material are removed by preliminary treatment with ribonuclease. Fibres comprising the diaphragm of the newborn rat display similar aggregations of subsarcolemmal ribosomes and peripheral basophilia. These ultrastructural and cytochemical manifestations of protein synthesis correspond closely in time to the well documented ‘supersensitivity’ to acetylcholine, which occurs along the entire length of muscle fibres in the newborn and denervated rat diaphragm. It is suggested, therefore, that this protein-synthetic machinery, which is concentrated at the cell surface, is involved in the formation of new receptors.
Surface modification and electrophoresis of normal and transformed BHK21 cells
Authors: Latner A. L. , Turner G. A.
Published: April 2021

The mean electrophoretic mobilities at pH 7·5 of virus-transformed (Py6) and normal BHK21 cells were very similar, whether they were harvested mechanically or by the use of trypsin. After formaldehyde treatment, there was significantly increased mobility in both cell types; the transformed cells showed significantly the greater change. After neuraminidase treatment, the mean electrophoretic mobility was decreased to the same extent in both types of cell. Treatment with neuraminidase and formaldehyde had no effect on the mean electrophoretic mobility of the normal cells but slowed the transformed variety. The mobility in histone solution had no relationship to histone concentration but was statistically correlated with the amount of histone per cell, calculated from total histone present divided by the total number of cells; a linear relationship being obtained with the normal cells but an initial plateau was demonstrated with the transformed cells. The normal cell line showed a similar plateau after neuraminidase treatment. The possible significance of these results is discussed.
The densities of colloidal iron hydroxide particles bound to microvilli and the spaces between them: Studies on glutaraldehyde-fixed ehrlich ascites tumour cells
Authors: Weiss L. , Subjeck J. R.
Published: April 2021

The densities of positively charged, colloidal iron hydroxide particles binding to the surfaces of glutaraldehyde-fixed cultured Ehrlich ascites tumour cells, were determined on cells maintained in suspension and monolayer cultures. In both cases, there was a higher density of particles bound to the surfaces of microvilli than to the spaces between them. Prior treatment of the cells with ribonuclease did not significantly alter the proportions of particles binding to the 2 different surface regions, indicating that the ribonuclease-susceptible binding sites are distributed with similar mean densities over the microvilli and the regions between them. Prior treatment of the cells with neuraminidase reduced the densities of particles binding to microvilli to those of the intermicrovillous spaces, indicating that the original higher density over the microvilli is due to an increased number of sialic acid residues at their surfaces.

The findings are discussed in relationship to observed changes in the surface density of microvilli when these cells convert from suspension to monolayer cultures.
Studies on the factors affecting cellular spreading upon culture-substrate: 1. the effects of divalent cations and conditioned medium on cellular spreading
Authors: Yasuda Kunio
Published: April 2021

The effects of various divalent cations on cellular spreading of in vitro cultured fibroblasts of chick embryos upon substrates were investigated in serum-free Eagle’s MEM. Fibroblasts spread on the surfaces of culture dishes in the presence of Mn2+, Mg2+, Ca2+ or Co2+, but not in the presence of Zn2+, Cd2+, Sr2+ or Cu2+. The minimum cation concentration for permitting cellular spreading at 37 °C is different for each cation. The values are 10−6, 5 × 10−4, 10−3 and 10−2 M for Mn2+, Mg2+, Ca2+ and Co2+, respectively. Below these concentrations the cells remain round. Mn2+ is about 1000-fold more effective in causing spreading than Ca2+. The cells do not spread at low temperature, but they do at 22 °C in the presence of 10−4 M Mn2+.

In a medium conditioned with a mass culture of fibroblasts, cell spreading occurs at one tenth of the cation concentrations given above. If conditioned medium is heated at 65 °C for 30 min or treated with trypsin, its effect is abolished; this suggests that the effective factor(s) in the conditioned medium is a protein.
Ultrastructural evidence for myosin of the smooth muscle type at the surface of trypsin-dissociated embryonic chick cells
Authors: Gwynn I. AP , Kemp R. B. , Jones B. M. , Gröschel-Stewart U.
Published: April 2021

Cells dissociated from embryonic chick muscle tissue using trypsin were rotated in the presence of globulin-enriched rabbit antisera against both sm∞th and striated muscle actomyosins originating from chicken gizzard (GAM) and pectoralis (PAM) muscles respectively. The presence of the rabbit antibodies was demonstrated using peroxidase-labelled sheep antirabbit γ-globulins, the enzyme-antibody conjugate being located by electron-microscope histochemistry.

Anti-GAM γ-globulins reacted strongly with the plasma membrane. Judging from the complete absence of staining, γ-globulins from non-immunized rabbit serum did not interact with the membrane. When γ-globulins of sheep anti-rabbit IgG serum were applied alone, that is in the absence of pretreatment with rabbit γ-globulin, there was an observable reaction with the cell surface. Preincubation of anti-GAM with the heavy meromyosin fraction from smooth-muscle myosin inhibited the interaction of the antibodies with the membrane, as evidenced by the absence of staining. A weak positive reaction obtained with anti-PAM was due to components of the antibody preparation which were reactive with actin and not with PAM.

It was concluded that a smooth-muscle myosin-like protein is an integral part of the plasma membrane of embryonic chick muscle cells.
Configuration of flagellar microtubule subunits
Authors: Warner F. D. , Meza I.
Published: April 2021

Microtubule protofilaments and their subunits isolated from sperm flagellar doublet tubules of the sea urchin Strongylocentrotus purpuratus were examined by analytical biochemistry and high-resolution negative staining electron microscopy. All microtubule (tubulin) fractions show 2 polypeptide bands (α and β tubulins) in an approximate 1:1 ratio on urea-polyacryl-amide gel electrophoresis. Heat (37 °C)-solubilized microtubules yield a protein fraction containing the tubulin dimer of molecular weight 115 000 Daltons. The dimeric tubulin subunit, as seen in the electron microscope, has an overall size of about 3·5 × 8 nm and appears to have the configuration of a figure 8 because of stain penetration into the centre of each of its 2 halves (figure os). Isolated protofilaments (3·5 ± 0·3 nm thick) can each be resolved into 2 subfilaments (1·7 ± 0·2 nm thick). The 2 subfilaments have periodic lateral associations resulting in the basic 4-nm subunit repeat (figure o) along the protofilament. Examination of collapsed and solubilizing protofilaments shows the figure 8 (dimeric) subunits separating at random along the proto-filaments. We conclude that the tubulin dimer must be composed of either elongated or bilobed monomers which result in the figure 8 configuration and hence the 2-stranded appearance of the protofilaments.
Characteristics of sea-urchin mitotic apparatus isolated using a dimethyl sulphoxide/glycerol medium
Authors: Forer A. , Zimmerman A. M.
Published: April 2021

A method for isolating sea-urchin zygote mitotic apparatus (MA) is described which is based on the Filner-Behnke method of isolating brain microtubules. MA were isolated in 50% (v/v) glycerol, 10% (v/v) dimethyl sulphoxide, 5 mM MgCl2, 0·1 mM EGTA, and 5 mM Sorensen’s phosphate buffer at a final pH of 6·8. MA stored at room temperature in isolation medium had stable birefringence, stable microtubules, and stable solubility properties (in 0·5 M KC1) over a period of 10 days to 2 weeks. These MA also seem to have more dry matter per volume than do MA isolated using hexylene glycol. The biggest disadvantages of the method are that zygotes often are difficult to lyse, and that cytoplasmic debris the same size as the MA sometimes contaminates the MA pellet.
Mitochondrial dna from 4-cell stages of Ascaris lumbricoides
Authors: Tobler H. , Gut C.
Published: April 2021

Mitochondrial DNA (mtDNA) has been isolated from 4-cell stages of Ascaris lumbricoides. This DNA amounts to about 40% of the total quantity of 4-cell-stage DNA. Its buoyant density in neutral CsCl gradients is 1·686 g cm” −3. Electron microscopy of mtDNA demonstrated the presence of circular molecules with an average contour length of 4·64 /<m. About 15% of these molecules are supercoiled, covalently closed circles, whereas some 2% consist of double-forked circular molecules. The form and size of these branched molecules suggest that they are replicative intermediates.
Histotypic cell aggregation in the presence of cytochalasin b
Authors: Maslow D. E. , Mayhew E.
Published: April 2021

A study was made of the effects of cytochalasin B on (a) specific sorting of reaggregating cells; (b) redistribution of cell types after treatment of preformed aggregates; and (c) the ability of aggregates of one cell type to incorporate and sort cells of another type. Freshly disaggregated neural retina and heart cells were cultured on a gyratory shaker at 70 rev/min and the aggregates formed analysed for sorting of cell types.

Cytochalasin B disrupted the sorting of forming aggregates at concentrations of 1 μg/ml and greater. The distribution of cell types in aggregates that were treated with cytochalasin after 24 h of culture was more random than the control.

Untreated cultures of retinal aggregates and heart cell suspension resulted in pure retinal and pure heart aggregates, but with more than 50% mixed and sorted aggregates. Cytochalasin B treatment resulted in fewer mixed aggregates and a higher proportion of pure retina and pure heart aggregates.
The effect of prolactin on the number of membrane-associated particles in kidney cells of the euryhaline teleost Gasterosteus aculeatus during transfer from seawater to freshwater: A freeze-etch study
Authors: Bonga S. E. Wendelaar , Veenhuis M.
Published: April 2021

The membranes of kidney cells of 3-spined sticklebacks were examined in freeze-etch replicas. The numbers of particles adhering to surfaces and fracture faces of the outer cell membranes and the membranes of the basal labyrinth were determined. The latter membranes probably are the main location of ion-transporting enzyme complexes.

The total number of particles per cell in freshwater fish exceeds that of seawater fish by about 50% for the outer cell membrane, and by almost 200% for the membranes of the basal labyrinth.

After transfer of seawater fish to freshwater, particle numbers increase and their densities approximate freshwater values after 20 h. This rise in particle numbers coincides with the increase of ion-transporting activity of the cells known to take place after transfer to freshwater.

The rate of increase of particle densities is enhanced after injection of ovine prolactin. This hormone is known to stimulate Na+/K+-ATPase activity of the basal labyrinth of teleost kidney cells.

The results indicate that the particles represent enzyme complexes. The number of particles is probably under hormonal control. The increase in particle densities after transfer to fresh water is accompanied by a rise in the number of nuclear pores, which is noticeable by 10 h.

No changes were observed in the density of the particles adhering to the fracture faces of gap junctions.
An electron-microscope study of the lampbrush chromosomes of the newt Triturus Cristatus
Authors: Mott Margaret R. , Callan H. G.
Published: April 2021

The ultrastructure of lampbrush chromosomes has been examined in sections of endembedded spread preparations, where the nuclear sap was dispersed prior to fixation, and in oocyte nuclei fixed entire, in 60-kV and i-MV electron microscopes. In spread preparations the axial chromomeres are seen to be organized as regularly spaced, unravelled skeins of DNP, each with a skein width of some 30 nm, though in some chromomeres there are regions where the DNP is much more densely packed. In both unravelled and dense regions, the ‘ultimate’ DNP fibres, wherever they can be identified, appear to be some 5 nm wide and thrown into loose coils. The unravelled state, although it clearly reflects an orderly packaging of the non-transcribing DNP, is an artifact of preparation; in sections of entire nuclei all chromomeres are seen to consist of DNP fibrils in the more densely packed state. The interchromeric fibril is single, and some 10 nm or less wide; it shows no sign of transcriptional activity.

In sections of end-embedded preparations the RNP matrix of most lateral loops, where transcription occurs, is seen to be made up of particles, each uniformly some 30 nm in diameter and strung together in linear array. These RNP particles are equally evident in sections of whole nuclei. In many loops the strings of particles are wound back on themselves to form regularly spaced, dense aggregates, each some 200–300 nm wide or wider; the larger aggregates can be resolved in the light microscope. The RNP particles are of the same dimensions throughout the lengths of individual lateral loops, and of substantially the same dimensions in loops of different gross morphologies. It is suggested that as each successive short length of RNA is transcribed from loop axis DNA, a protein associates with this RNA and winds it up to form a ‘manageable’ package, allowing transcription to proceed.
Colchicine resistance in mammalian cell lines
Authors: Minor P. D. , Roscoe D. H.
Published: April 2021

Colchicine-resistant variants derived from mouse and Syrian hamster lines are described. The resistant cells do not appear to be true mutants, since they appear at a high frequency, unaffected by treatment with ethyl methyl sulphonate, and are unstable in the absence of the drug. They are cross-resistant to other drugs, show a reduced rate of binding of colchicine in monolayer, and give extracts with colchicine-binding properties identical to those of the wild type. Thus the resistance is due to a permeability barrier. The naturally occurring resistance of the Syrian hamster line is specific for colchicine, and may be due to a specific permeability barrier. The Syrian hamster line is also shown to have an extra colchicine-binding pool.
The fine structure and cell kinetics of mouse epidermis after wounding
Authors: Potten C. S. , Allen T. D.
Published: April 2021

A variable amount of cornified tissue removed from mouse dorsal epidermis results in stimulation of the entire basal layer. Stimulation does not appear to be dependent on damage to an indiviaual epidermal proliferative unit (EPU). The immediate reaction to wounding is a rapid movement of cells from the basal layer to the differentiating compartment resulting in depopulation of the basal layer, which is followed by a burst in DNA-synthetic activity. The result of the increased transit of cells through the epidermis is that various aspects of keratinization can appear abnormal. The Langerhans cells show several changes, often appearing suprabasal and becoming smaller, rounded cells with a less-clear cytoplasm and fewer granules. The initial migratory reaction results in a largely normal epidermis on the third day. This reaction is followed by a transient hyperplasia which reaches its peak on the sixth to seventh day and gradually returns to normal by the fourteenth to fifteenth day. The hyperplasia is characterized by a loss of the ordered stacking of cornified cells which become shorter and thicker than normal. There is a return to the stacked state beginning on the tenth day. The Langerhans frequency is apparently at its lowest on days 6â€“7 when the proliferation levels are at their maximum. An inverse relationship appears to exist between relative Langerhans cell frequency and cell proliferation rate. The data suggest that the frequency of Langerhans granules also changes during the course of the hyperplasia, peak levels being observed just before the decline in proliferative activity.
Inhibition of rrna synthesis following incorporation of 5-bromodeoxyuridine into dna of Tetrahymena pyriformis
Authors: Lykkesfeldt Anne E. , Andersen H. A.
Published: April 2021

Tetrahymena pyriformis was grown on chemically defined medium in the presence of 5-bromodeoxyuridine (BUdR). Under these growth conditions more than 60% of the thymidine sites in DNA were substituted with BUdR. It was found that RNA synthesis was strongly inhibited by the presence of BUdR in. DNA. To assure that incorporation of BUdR into DNA was a prerequisite of the effect observed, BUdR was added to synchronously dividing cells. BUdR had no effect on the cells when present outside the period of nuclear DNA replication, whereas RNA synthesis was strongly inhibited as soon as the genes coding for ribosomal RNA had replicated in the presence of BUdR.
Dna synthesis in growth and encystment of Acanthamoeba castellanii
Authors: Roti L. W. Roti , Stevens A. R.
Published: April 2021

Differentiation of Acanthamoeba castellanii into dormant cysts occurs spontaneously in stationary phase cultures, or can be induced experimentally by starvation. Although no further increase in cell density occurred after induction in either case, incorporation of [3H]thymidine into DNA continued at a reduced rate through the period when differentiated products (cyst wall components) were formed. No net accumulation of DNA occurred during differentiation, indicating that the DNA synthesis occurring at this time was balanced by breakdown.

When either 5-fluorodeoxyuridine (FUdR) or hydroxyurea was added to exponentially growing cultures, growth was terminated and the subsequent spontaneous encystment was delayed in comparison with untreated stationary phase cultures. A similar delay was observed for experimentally induced encystment of FUdR-pretreated cells. In all cases, delay of encystment was correlated with inhibition of 32PO4 incorporation into DNA, and unexpectedly also into RNA. Addition of FUdR at zero-time of experimental induction of cells not previously exposed to FUdR, on the other hand, had no effect on encystment or on “PO4 incorporation. The delay of encystment produced by FUdR and hydroxyurea, therefore, appeared to reflect a requirement for normal synthesis of DNA and/or RNA not only during encystment, but also during the period of exponential growth just before encystment induction.
Changes in the organization of chromosomes during the cell cycle: Response to ultraviolet light
Authors: Schor S. L. , Johnson R. T. , Waldren C. A.
Published: April 2021

Fusion between mitotic and interphase cells results in the premature condensation of the interphase chromosomes into a morphology related to the position in the cell cycle at the time of fusion. These prematurely condensed chromosomes (PCC) have been used in conjunction with u.v. irradiation to examine the interphase chromosome condensation cycle of HeLa cells. The following observations have been made:

(1) There is a progressive decondensation of the chromosomes during G1 which is accentuated by u.v. irradiation:

(2) The chromosomes become more resistant to u.v.-induced decondensation during G1 and mitosis.

(3) There is a close correlation between the degree of chromosome decondensation and the amount of unscheduled DNA synthesis induced by u.v. irradiation during G1 and mitosis:

(4) Hydroxyurea enhances the ability of u.v. irradiation to promote the decondensation of chromosomes during G1, G1 and mitosis. Hydroxyurea also potentiates the lethal action of u.v. irradiation during mitosis and G1.

These data are discussed in relation to the suggestion that chromosomes undergo a progressive decondensation during G1 and condensation during G2.
Retention of endocrine function by an insulin-secreting pancreatic islet cell tumour from syrian hamster through serial transplantation in Nude mice
Authors: Shin Seung-Il , Baum S. G. , Fleischer N. , Rosen O. M.
Published: April 2021

An insulin-secreting islet cell tumour of the Syrian hamster has been transplanted serially in the congenitally immune-deficient nude mouse, in order to test the potential usefulness of this mouse mutant as a graft carrier of heterologous tumours with stable differentiated phenotypes. The incidence of tumour growth was very high, and the hamster tumour retained its functional and histologic characteristics during consecutive passages in nude mice. These results show that nude mice may be useful carriers of differentiated tumours from non-inbred species including man, and for the isolation of cell lines from such tumours.
Adhesion of red blood cells to charged interfaces between immiscible liquids. a new method
Authors: Gingell D. , Todd I.
Published: April 2021

We have devised a method of making a flat oil/water interface which remains flat on inversion. Cell adhesion to the interface can be observed microscopically.

Glutaraldehyde-fixed human red blood cells adhere to the interface between physiological saline and hexadecane containing surface-active behenic acid at pH values below about 7·5. At high pH values, cells are prevented from adhering due to dissociation of the carboxyl groups of behenic acid oriented in the interface. The negative red cells are driven away electrostatically.

Adherent and non-adherent cells remain on the aqueous side of the interface and do not appreciably deform it when adherent. Cells are electrostatically attracted to a similar interface containing positively charged octadecyltrimethylammonium ions. Cells also adhere to an interface containing octadecanol, which carries no charge.

Underlying both electrostatic repulsion and attraction between red cells and oil/water interfaces is an attractive force which may be of electrodynamic (van der Waals) origin.
Cell movement and adhesion in the developing chick wing bud: studies on cultured mesenchyme cells from normal and Talpid3 mutant embryos
Authors: Ede D. A. , Flint O. P.
Published: April 2021

Mesenchyme fragments from early wing buds of normal and talpid3 mutant chick embryos were explanted for culture in plastic Petri dishes and the behaviour of individual cells as they moved out on to the plastic surface was studied by time-lapse ciné photography, followed by statistical analysis. Two parameters of cell movement were recorded: (1) the distances moved over measured 100-s intervals and (2) the length of time each cell spent at rest before moving on. The average speed of movement over the whole path tracked for each cell, inclusive of time at rest, was significantly greater in normal than in talpid3 cells. There was no significant difference between normal and mutant cells in the average distance moved per 100-s step, equivalent to the speed over the whole path exclusive of time at rest, but the percentage of time spent at rest was significantly less in normal than in talpid3 cells. This difference appears to be related to a difference in cell morphology, since it was observed that the mutant cells were more flattened than normals, with very extensive ruffled membranes and short spiky microvilli all round the cell periphery. The relation of these differences in cell morphology and behaviour in vitro to the production of the characteristically fan-shaped limb bud outgrowth and altered pattern of cartilage elements in the developing mutant limb bud is discussed.
Cell surface lipids and adhesion: III. the effects on cell adhesion of changes in plasmalemmal lipids
Authors: Curtis A. S. G. , Chandler C. , Picton N.
Published: April 2021

The two preceding papers of this series suggest that the state of the plasmalemmal lipids affects cell adhesion. Plasmalemmal composition was altered by the experimental incorporation of fatty acids into R1 and R2 positions in the phosphatidyl components of the cell surface. In this paper we report that: If the incorporation is of long chain length fatty acids (saturated) cell adhesion rises.If the incorporation is of unsaturated fatty acids cell adhesion falls as the unsaturation increases.Incorporation has to be extensive to produce a large change in adhesion.Changes in adhesion parallel the plasmalemmal incorporation but do not follow the total cell incorporation.

Item (4) argues that it is plasmalemmal and not other membrane lipids that are involved in cell adhesion. Item (3) suggests that bulk membrane properties and not some very specific grouping are involved in the effects of lipids on adhesion. The similar extents of incorporation of the various different fatty acids and the negligible amounts of lysophospholipids in the membranes of cells that have incorporated fatty acids argue that the effects are not due to differential accumulations of these lysolipids when incubations are done with different fatty acids. The changes in adhesion cannot be accounted for by changes in surface charge density since the electrophoretic mobility of the cells is unchanged by these incubations.

It is suggested that these effects on adhesion due to changes in plasmalemmal lipids can be explained either in terms of the action of intermembrane van der Waals-London (electrodynamic) forces in cell adhesion or of changes in surface fluidity. These alternatives are discussed.
The induction of dna synthesis in the chick red cell nucleus in heterokaryons during the first cell cycle after fusion with hela cells
Authors: Johnson R. T. , Mullinger A. M.
Published: April 2021

Induction of DNA synthesis in embryonic chick red cells has been examined during the first and second cell cycles after fusion with HeLa cells synchronized in different parts of G1 and S-phase. The data indicate that: (i) the younger the embryonic blood the more rapidly the red cells are induced into DNA synthesis; (ii) the greater the ratio of HeLa to chick nuclei in the heterokaryon, the more rapidly the induction occurs; (iii) DNA synthesis in the chick nucleus can continue after the HeLa nucleus has left S-phase and entered either G2 or mitosis; (iv) the induction potential of late S-phase HeLa is somewhat lower than that of early or mid S-phase cells; (v) less than 10 % of the chick DNA is replicated during the first cycle after fusion and only a small proportion (15 %) of the chick nuclei approach the 4C value of DNA during the second cycle after fusion; (vi) the newly synthesized DNA is associated either with the condensed regions of the nucleus or with the boundaries between condensed and non-condensed regions; (vii) the chick chromosomes at the first and second mitoses after fusion are in the form of PCC (prematurely condensed chromosomes); they are never fully replicated and are often fragmentary; (viii) DNA synthesis in the chick nucleus is accompanied by an influx of protein (both G1 and S-phase protein) from the HeLa component of the heterokaryon.
The growth of the pollen tube wall in Oenothera organensis
Authors: Dickinson H. G. , Lawson Janice
Published: April 2021

The growth of the pollen tube wall of Oenothera is effected by the expulsion of fibrillar material from the cytoplasm into the developing wall. This material may also be seen in the cytoplasm, contained in membrane-bound vesicles. It is not clear how the content of the vesicles is discharged, but it appears not to involve the participation of microtubules.

The source of the cytoplasmic fibrillar bodies depends upon the stage of development of the pollen tube. The earliest growth is derived from the inclusion into the wall of vesicles containing pre-formed materials present in the grain on pollination. During the next stage of growth the wall is derived from the content of double-membraned inclusions also present in the pollen. The content of the former vesicles is not so similar to the wall as the latter, but intermediates between the 2 types of vesicle may be seen in the cytoplasm, indicating that the former are formed from the latter. Most of the tube wall is derived from the products of dictyosomes in the pollen grain or tube. These dictyosomes are few in number and they must be exceedingly active. This, and the observation that dictyosome vesicles are frequently associated with banked complexes of mitochondria, indicates that some steps in the metabolism of the vesicular content, perhaps phosphorylation, take place distant from the dictyosomes.

These different sources of fibrillar material presumably permit the rapid starting of tube growth, without any attendant metabolism. However, it would be impossible to include enough pre-formed wall material in the grain to enable the full growth of the tube, so once started, it seems that the tube then relies on the elaboration of simple reserves for the construction of its wall. These reserves are likely to be held in the pollen, and may be the large numbers of starch grains characteristic of the pollen cytoplasm.
Hepatocyte function in long-term organ culture of Amphiuma means liver
Authors: Fleming N. , Brown D. , Balls M.
Published: April 2021

Fragments of liver from the adult urodele Amphiuma means, the Congo eel, were maintained in organ culture for up to 70 days. The normal electrophoretic patterns of several enzymes were retained. The activities of ornithine transcarbamylase, arginase, glutamate oxalacetate transaminase and glutamate pyruvate transaminase, and urea production, glucose uptake and tissue glycogen content remained relatively constant throughout the culture period. Histological organization and hepatocyte ultrastructure were also retained. Liver fragments survived better in media based on MEM or BME than in medium based on Leibovitz L15. Since many aspects of tissue-specific structure and function are retained, long-term amphibian organ culture is well suited to studies on the control of hepatocyte function and on the effects of metabolites, hormones, drugs and toxins.
An electron-microscope study of the intranucleolar chromatin during nucleologenesis in root meristematic cells ofAllium cepa
Authors: Chouinard L. A.
Published: April 2021

The various states of condensation of the chromatin material, contained inside the lacunar regions of the reforming nucleolus in Allium cepa, have been investigated by means of conventional electron-microscope techniques. The observations reveal that, in the emerging early to late telophase nucleoli, the intralacunar chromatin material in question appears both in an extended and a condensed condition; from late telophase to the mid G1 period of interphase, the intralacunar chromatin material of the rapidly growing and developing nucleoli is present in an extended state only.

An attempt is made to interpret these morphological findings in the light of current knowledge concerning the structural relationship of the nucleolar organizing region of the nucleolar chromosome with the interphase nucleolus in plant cells. The relevant observational evidence would be consistent with the view that the chromatin-containing lacunar regions of the reforming nucleolus in Allium cepa correspond, in fact, to cross- or oblique sections of a meandering channel through which the nucleolar organizing segment of the nucleolar chromosome passes. Assuming the applicability to intranucleolar chromatin of the general concept of condensed-inactive versus extended-active chromatin, it is concluded that gradual uncoiling and subsequent decondensation of the chromatin of the nucleolar organizing region in the form of a convoluted loop structure are key morphological and functional events associated with the process of nucleologenesis in the species investigated.
Spontaneous and lectin-induced redistribution of cell surface receptors on embryonic chick neural retina cells
Authors: Mcdonough J. , Lilien J.
Published: April 2021

The mobility of plant lectin receptors in the plane of the membrane is examined for cells prepared from embryonic chick neural retinas by a variety of procedures. Cells liberated from the intact tissue by trypsin treatment followed by mechanical dissociation are able to redistribute their receptors into ‘caps ‘both spontaneously and in the presence of a multivalent lectin. These cells, dispersed by trypsinization, upon repair in culture for a suitable period of time lose their ability to redistribute lectin receptors. Cells dispersed by mechanical means without prior trypsin treatment are unable to undergo ‘cap’ formation. In addition, cells within intact tissues are also unable to redistribute their lectin receptors into ‘caps. ‘Based on these observations we propose that within solid tissues which have assumed their characteristic architecture, cell surfaces are immobilized, and that this phenomenon may be a critical parameter in determining the potential of a cell to undergo morphogenetic rearrangements.
Fine structure of the nucleolus in normal and mutant Xenopus embryos
Authors: Hay Elizabeth D. , Gurdon J. B.
Published: April 2021

Mutant and normal Xenopus laevis embryos (o-nu, 1-nu, 2-nu) were examined in the electron microscope after glutaraldehyde and/or osmium-tetroxide fixation. During cleavage both 0-nu and wild-type embryos contain multiple small nucleolar bodies, less than 1 μ in diameter, composed mainly of a fibrous material. By the end of cleavage or beginning of gastrulation, granular caps develop on the fibrous nucleolar bodies. In 1- and 2-nu cells, the multiple nucleolar bodies are replaced during gastrula and neurula stages by definitive nucleoli (2–5 Å in diameter) which contain abundant small (150 Å) granules intermingled with fibrous material. In o-nu cells, one or two pseudonucleoli (1–3 μ in diameter) appear at about the same time that definitive nucleoli develop in wild-type cells. The multiple small nucleolar bodies disappear as the pseudonucleoli enlarge. Pseudonucleoli differ from definitive nucleoli in having a much smaller amount of the granular component, which is located as a cap on the periphery of the fibrous component and not mingled with it. The granular component of the 0-nu pseudonucleoli, however, is not distinguishable in its fine structure from the same component of normal nucleoli. In many o-nu tadpoles at stage 41, the granular component of the nucleolus is entirely absent and the fibrillar component is very prominent. Both granular and fibrous components of the o-nu pseudonucleoli contain RNA as judged by RNase sensitivity and staining affinity for basic dyes.
A study of mitochondrial membranes in relation to elementary particles
Authors: Malhotra S. K. , Eakin R. T.
Published: April 2021

Elementary particles that commonly have been seen by electron microscopy to be attached by stalks to mitochondrial cristae in negatively stained preparations, were not apparent in similarly stained mitochondria from exponentially growing wild-type Neurospora crassa when these were isolated in sucrose solution containing 1 × 10−3 M EDTA. However, elementary particles were easily demonstrable in electron micrographs if the mitochondria were isolated without EDTA in the sucrose solution. A biochemical study indicated that both kinds of mitochondrial preparations, isolated in the presence or absence of EDTA, had about the same capacity for oxidative phosphorylation. Observations on rat-liver mitochondria also suggested that the stalked elementary particles were more easily demonstrated if the preparation was made in the absence of EDTA. It was difficult to demonstrate elementary particles in wild-type Neurospora mitochondria isolated with or without EDTA and subsequently prepared for electron microscopy by spreading on the surface of an aqueous solution of potassium phosphotungstate. Elementary particles could be demonstrated in poky Neurospora mitochondria isolated with EDTA if the mitochondria were spread on the surface of an aqueous solution of phosphotungstate. It was concluded that biochemical functions associated with elementary particles are independent of structural configuration as seen by electron microscopy.
Factors affecting the tertiary structure of resilin in locusts
Authors: Neville A. C.
Published: April 2021

Autofluorescent daily growth zones in locust resilin provide a detailed record of cross-linking of the polypeptide chains, since the fluorescence is caused by the tertiary cross-links, dityrosine and trityrosine. Environmental temperature changes are the main cause of autofluorescent daily growth layers in resilin, fluorescence increasing with temperature. Microdensitometric scans show that this rhythmicity is superficial, in harmony with the optical and mechanical isotropy of resilin. The molecular structure and properties of resilin appear to be homeo-statically controlled by the cells. With suitable rearing conditions, resilin ligaments were produced with half or twice as many fluorescent growth layers as usual, or with them almost absent. Evidence for a weak circadian rhythm influencing cross-linking is presented. A marked drop in fluorescence followed by a sharp rise occurs at adult emergence and is metabolically triggered. A metabolic control also operates to make the resilin grown before the moult less fluorescent than resilin grown after it. Further evidence for the very rapid cross-linking of resilin during its formation is discussed.