bluuebunny/crossref_metadata_2025 huggingface dataset:
The glucose, insulin and glutamine requirements of suspension cultures of hela cells in a defined culture medium
Authors: Blaker G. J. , Birch J. R. , Pirt S. J.
Published: April 2021

The serum supplement in a defined medium for the growth of HeLa cells could be replaced by protamine-zinc-insulin (0·2 u./ml). Insulin (0·4 u./ml) replaced the growth-stimulatory properties of protamine-zinc-insulin, whilst protamine sulphate (5 μg/ml) was found to be toxic to the cells. The addition of insulin to cultures depleted of insulin increased both cell growth rates and maximum cell populations. In the defined medium, HeLa cells could only utilize glutamate when a small amount of glutamine was included. Glucose, at a level of 2 mg/ml, was shown to limit maximum cell populations. The growth yield from glucose was 295 μg cell dry weight/mg glucose. When the medium glucose concentration was increased to 4 mg/ml, HeLa cell populations in excess of 16×105 cells (i.e. 640 μg dry weight)/ml were routinely achieved in the defined medium supplemented with insulin. Growth is then limited by the amino acid supply. Increasing the amino acid concentration of the medium by 50% raised the maximum cell population to 23·5 × 105 cells (i.e. 940 μg dry weight)/ml.
Fine structure of swarmers of Cladophora and Chaetomorpha: I. the plasmalemma and golgi apparatus in naked swarmers
Authors: Robinson D. G. , Preston R. D.
Published: April 2021

Naked swarmers of both Cladophora rupestris and Chaetomorpha melagonium have been examined by the freeze-etching technique. The swarmers of Cladophora, collected just after settling, reveal several layers of granules external to the plasmalemma and internal to the so-called ‘fibrous-layer’. Chaetomorpha swarmers collected just before settling show extrusion of vesicles through the plasmalemma. The structures associated with the membranes are discussed in relation to known features of these swarmers already observed by sectioning. The role of granules in the synthesis of cell wall microfibrils is strengthened though the spatial arrangement of the granules seen in this investigation does not completely fulfil the ‘ordered granule’ hypothesis.

Description of, and comments on, features related to cell wall synthesis, particularly the Golgi and vacuolar systems, are given.
Synaptic structures in the nuclei of sporulating yeast, Saccharomyces cerevisiae (hansen)
Authors: Moens P. B. , Rapport E.
Published: April 2021

After 4 h on sporulation medium the larger cells have formed, within the nucleolus, a spherical body of amorphous substance which stains less densely than the nucleolus. At 8 h most of these bodies contain synaptonemal complex-like structures. There is usually only one such body per nucleus. Only rarely are normal synaptonemal complexes detectable in the nucleus. At the first meiotic division these bodies are still present but they no longer have polycomplexes associated with them. At this time they become sequestered in a nuclear evagination and they are no longer detectable after the second meiotic division. It is argued that the polycomplex body may elaborate synaptic elements which function in meiotic chromosome pairing but that the resulting complexes are difficult to detect because of the lack of chromosome condensation, a characteristic of several fungi.
Growth and changes in pool and macromolecular components of Schizosaccharomyces pombe during the cell cycle
Authors: Stebbing Nowell
Published: April 2021

Amino acids, nucleotide and carbohydrate material were found to account for 46 % of the total dry weight of pool material in Schizosaccharomyces pombe growing in minimal medium. The composition of the amino acid pool was also determined by autoanalysis and was found to be unaltered during growth in 2 M sorbitol, indicating that pool amino acids are not important in the osmoregulation of the cell. Kinetic analysis of the amino acid pool using 14C-labelled amino acids showed that amino acids accumulated from the medium enter an ‘expandable’ pool distinct from the ‘internal ‘pool which is maintained during growth on minimal medium.

Total RNA, protein, pool amino acid and pool ‘nucleotide’ material were estimated in synchronous cultures grown in minimal medium. All these components appeared to accumulate in an exponential manner during the cell cycle. Direct estimation of total cellular dry weight and the total pool in synchronous cultures showed that total cell dry weight increased exponentially and the pool did not fluctuate during growth in minimal medium. This contrasts with previous work on single cells of S. pombe grown in complex medium which showed that the dry weight of the pool fluctuates during the cell cycle and total cell dry weight increased linearly. Linear growth of S. pombe in malt extract broth can be accounted for by the presence of the second (‘expandable ‘) pool of amino acids formed during growth in complex medium.

The phenomenon of linear growth during the cell cycle is shown to occur generally only in cells growing in complex medium. The phenomenon is considered in relation to mechanisms for controlling the size of the pool during growth in complex media.
Identification of a set of calcium-binding proteins in reticuloplasm, the luminal content of the endoplasmic reticulum
Authors: Macer Darryl R. J. , Koch Gordon L. E.
Published: April 2021

A procedure was developed for the isolation of reticuloplasm, the luminal material of the endoplasmic reticulum (ER). A reticuloplasm-rich extract was prepared from a murine plasmacytoma cell line that contains large amounts of ER, by first extracting the cytoplasmic contents using hypotonic lysis to yield ER-rich ‘shells’ followed by mechanical lysis to release the ER contents. The extract contains five major proteins with apparent molecular weights of 100, 75, 60, 58 and 55 (×103)Mr by SDS-polyacrylamide gel electrophoresis. The 100, 75 and 58 (×103)Mr species were identified as the known ER proteins endoplasmin, BiP and PD1, respectively. The ER association of the 60 and 55 (×103) Mr proteins was confirmed by confocal fluorescence microscopy with affinity-purified antibodies.

Equilibrium dialysis with isolated reticuloplasm gave a calcium-binding capacity of 300 nmoles calcium per mg protein with half-maximal binding at 3 mM-Ca2+. Purified endoplasmin bound 280 nmoles calcium per mg protein at a calcium concentration of 5 mM-Ca2+.

A calcium overlay test revealed that, in addition to endoplasmin, reticuloplasm contained at least three other calcium-binding proteins: i.e. BiP, PDI and the 55×103Mr protein, respectively, with endoplasmin and the 55×103Mr protein (CRP55) accounting for the major proportion of the calcium-binding activity.

Treatment of cells with calcium ionophore led to the specific over-expression of the major calcium-binding reticuloplasmins endoplasmin, BiP and CRP55.

These studies show that the lumen of the ER contains a family of proteins with the capacity to bind significant amounts of calcium in the millimolar range and thereby to confer upon the ER the ability to perform a calcium storage function analogous to that of the sarcoplasmic reticulum in muscle cells.
Spontaneous changes in intermediate filament protein expression patterns in lung cancer cell lines
Authors: Broers Jos L. V. , Rot Mirjam Klein , Oostendorp Tineke , Bepler Gerold , Leij Lou De , Carney Desmond N. , Vooijs G. Peter , Ramaekers Frans C. S.
Published: April 2021

The usefulness of cell lines in the study and prediction of the clinical behaviour of lung cancer is still a matter of debate. However, lung tumour cell cultures have been of value in investigations concerning molecular and cell biological aspects of these neoplasms. Especially in the examination of characteristics specific for the main types of differentiation (squamous cell carcinoma, adenocarcinoma, small cell carcinoma), in vitro studies have been most important.

Twenty eight lung cancer cell lines were cultured for up to four years, and were examined at regular intervals for their intermediate filament protein (IFP) expression patterns using a panel of cytokeratin (CK) and neurofilament (NF) antibodies. These studies showed that the classic type of small cell lung cancer (SCLC) cell lines contain CKs 8,18, and occasionally CK 19, while the variant-type SCLC cell lines generally express no CKs but can contain NFs. Non-SCLC cell lines, such as squamous cell carcinoma and adenocarcinoma cell lines, contain CKs 7 (in most cases), 8, 18 and 19. In one variant SCLC cell line and in one adenocarcinoma cell line CKs 4, 10 and 13, characteristic of squamous cell differentiation, were found. Although most cell lines have remained stable with respect to growth characteristics and IFP expression patterns, five lung cancer cultures exhibited a transition from one cell type to another, paralleled by changes in IFP expression. Progressions from classic to variant SCLC cell lines have been observed, next to conversions from variant SCLC to cell lines re-expressing cytokeratins. In some cases this resulted in a coexpression of CKs and NFs within a cell line and even within individual tumour cells. These results strongly support the earlier finding that CK expression in SCLC cell lines is a reliable marker for the classic type of differentiation, while the absence of CKs and the presence of NFs marks the variant type of differentiation. Our results are discussed in view of previous histological findings.
Characterization of monoclonal antibodies to trichocyst antigens in Paramecium
Authors: Fok Agnes K. , Olegario Rhodora P. , Ueno Marilynn S. , Allen Richard D.
Published: April 2021

Ten mouse monoclonal antibodies (mAbs) were raised against trichocyst contaminants present in crude or enriched lysosome fractions of Paramecium multimicronucleatum. Using an indirect immunofluorescence assay (IFA) and immunogold labelling on frozen thin sections, epitopes were located on the outer edge, cortex and the core of the trichocyst body, as well as the sheath covering the tip. Except for the two on the tip, epitopes were reactive after SDS-PAGE under non-reducing conditions. Four mAbs (131C1E8, Al–3, A16–2, D7) were directed to a trio of bands of 37, 34 and 29 (× 103) Mr from the beaded or meshlike trichocyst body sheath. A fifth mAb (135B9E7), directed to epitopes on the cortex inside the beaded body sheath, reacted strongly with the 37 and 34 bands, but weakly with the 29×103Mr band. The last three mAbs (270D5, 22C7F2, D8) were reactive with one or more of three families of antigens found on the trichocyst core. mAb 270D5 reacted mainly with the 34 and 29(×103)Mr bands of the family containing the above trio, while mAb 22C7F2 reacted consistently with the 47×103 band of the higher Mr family but variably with both the trio of bands and the 17×103 band of the lower Mr family. mAb D8, which was directed to epitopes on the trichocyst core and small vesicles in the endoplasm, reacted only with the 29×103Mt band. The mAbs were cross-reactive with the trichocysts of P. primaurelia, P. tetraurelia, P. caudatum and P. calkinsi with some small variation in blotting patterns. Except for the reactivity of mAbs 22C7F2 and 270D5 with a 64×103Mr band, Tetrahymena thermophila was unreactive with these mAbs either by IF A or after SDS–PAGE and blotting. These results show that: (1) the mAbs obtained are specific for selected polypeptides in four trichocyst locations; (2) antigenic differences exist within the core, cortex and the beaded sheath of the trichocyst body; and (3) the antigenic determinants were conserved in all paramecia species tested.
Chicken histone h5 inhibits transcription and replication when introduced into proliferating cells by microinjection
Authors: Bergman Mathias G. , Wawra Edgar , Winge MÅrten
Published: April 2021

Chicken erythrocyte histone H5 has been suggested repeatedly to be a general suppressor of transcription and replication. Therefore, the biological functions of H5 were investigated and compared with those of Hl (Hla + Hlb) by microinjection of the purified proteins into proliferating L6 rat myoblasts. By pulse-labelling of the injected cells with [3H] uridine and [3H] thymidine it was shown that H5 blocked both transcription and replication substantially, and that the chromatin of the injected cells became densely compacted. Hl also suppressed these functions, but to a much lesser degree. The effects were specific and not caused by change in intracellular pH caused by introduction of the very basic H5, or its non-specific interaction with nucleic acid, since injection of protamine or lysozyme did not affect the cells. The migration and localization of injected H5 was monitored at different times after injection by immunofluorescence, which revealed that H5 was efficiently and stably concentrated in the nucleus.

The results indicate that H5 indeed might function as an inactivator of the erythroid genome in its natural environment, probably by keeping the chromatin in a very condensed state.
Variation in cytoplasmic microtubule organization and spindle length between the two forms of the dimorphic fungus Candida albicans
Authors: Barton Richard , Gull Keith
Published: April 2021

Candida albicans is a dimorphic fungus capable of growing as a budding yeast and as a filamentous hypha. We have used the technique of immunofluorescence to study the changes in the microtubule cytoskeleton during the cell cycle in both growth forms. This approach has revealed the presence of an extensive system of microtubules, including cytoplasmic microtubules and a rod-like intranuclear spindle. We have provided a complete description of the arrangement of cytoplasmic and spindle microtubules at each phase of the yeast cell cycle. A novel and characteristic feature of the yeast phase of Candida is the presence of an array of short microtubules at the neck of the doublet cell. This neck-associated array (NAA), is apparently organized independently of the main microtubuleorganizing centre, the spindle pole bodies, at late anaphase. Analysis of microtubule patterns in the hyphal state reveals that the general arrangements of microtubules are similar to those seen in the yeast phase. These patterns suggest a role for the cytoplasmic microtubules in the nuclear migration that occurs during hyphal growth. A major finding is that the mitotic spindle in hyphae is considerably longer (12–20μm) than the spindle in yeast cells (7–8 μm). This may reflect the role of the hyphal mitotic spindle not only in nuclear division but also in the positioning of the daughter nuclei at the centres of hyphal compartments.
Expression pattern of two related cystic fibrosis-associated calcium-binding proteins in normal and abnormal tissues
Authors: Wilkinson Maureen M. , Busuttil A. , Hayward Caroline , Brock D. J. H. , Dorin Julia R. , Heyningen Veronica Van
Published: April 2021

This paper reports further study of the identity and function of a protein shown to be elevated in serum from cystic fibrosis (CF) patients and clinically normal heterozygotes. Monoclonal antibodies, specifically recognizing the tentatively named cystic fibrosis antigen (CFAg), were produced. Immunoaffinity purification of CFAg from several sources revealed two components: 11×103 and 14×103Mr proteins. cDNA clones corresponding to each protein have been isolated. Data-base comparisons of the deduced amino acid sequences suggest that both genes encode related but distinct calcium-binding proteins. We propose the name calgranulin A and B, for the 11×103 and 14×103Mr components, respectively. It is clear from the assignment of the calgranulin genes to chromosome 1 that neither is the product of the mutant CF gene, which maps to chromosome 7.

We have used the monoclonal antibodies to study the tissue distribution of the two proteins in a wideranging immunohistological survey. Where possible the pattern of expression was confirmed by RNA blot analysis. Strong calgranulin expression in granulocytes was confirmed. In addition to myeloid cells, a restricted subset of normal stratified squamous epithelia were found to be calgranulin-positive. These included tongue, oesophagus and buccal cells, the last of which has been shown to have altered calmodulin activity in CF patients. Using indirect alkaline phosphatase staining, tissue sections of lung, pancreas and skin (normally considered sites where the CF defect is expressed) were not calgranulin-positive. However, by indirect immunofluorescence, nasal polyp sections showed weak patchy calgranulin expression in some epithelial cells, and stronger, higher frequency expression when such cells were briefly cultured.

A number of hyperproliferative, neoplastic or frankly malignant epithelia were found to express the two proteins. Calgranulin expression is a good marker for ‘reactive’ epithelium in skin and for squamous cell carcinomas of skin, lung and buccal tissues. The calgranulin-positive permanent cell line from a buccal squamous cell carcinoma may prove a suitable tool for unravelling the calgranu-lin-CF relationship.
Dictyostelium discoideum mutant Synag 7 with altered g-protein–adenylate cyclase interaction
Authors: Snaar-Jagalska B. Ewa , Haastert Peter J. M. Van
Published: April 2021

Previous results have shown that Dictyostelium discoideum mutant synag 7 is defective in the regulation of adenylate cyclase by receptor agonists in vivo and by GTPγS in vitro; the guanine nucleotide activation of adenylate cyclase is restored by the high-speed supernatant from wild-type cells.

Here we report that in synag 7 membranes: (1) cyclic AMP receptors had normal levels and were regulated by guanine nucleotides as in wild-type; (2) GTP binding and high-affinity GTPase were reduced but still stimulated by cyclic AMP; (3) the supernatant from wild-type cells restored GTP binding to membranes of this mutant, and partly restored high-affinity GTPase activity; (4) the supernatant of synag 7 was ineffective in these reconstitutions and did not influence GTP binding and GTPase activities in mutant or wild-type membranes.

These results suggest that the defect in mutant synag 7 is located between G-protein and adenylate cyclase, and not between receptor and G-protein. A factor in the supernatant is absent in synag 7 and appears to be essential for normal GTP binding, GTPase and activation of adenylate cyclase. This soluble heat-labile factor may represent a new molecule required for receptor-and G-protein-mediated activation of adenylate cyclase.
MSH inhibits growth in a line of amelanotic hamster melanoma cells and induces increases in cyclic AMP levels and tyrosinase activity without inducing melanogenesis
Authors: Slominski Andrzej , Moellmann Gisela , Kuklinska Elizabeth
Published: April 2021

In Bomirski Ab amelanotic hamster melanoma cells, L-tyrosine and/or L-dopa induce increases in tyrosinase activity as well as synthesis of melanosomes and melanin. L-tyrosine also modifies melanocyte-stimulating hormone (MSH) binding. In this paper we show that in the Bomirski amelanotic melanoma system MSH and agents that raise inUa-cellular cyclic AMP induce dendrite formation, inhibit cell growth, and cause substantial increases in tyrosinase activity without inducing melanin synthesis. Tyrosinase activity is detected only in broken cell preparations, or cytochemically in fixed cells. In the continued absence of mature melanosomes, the induced enzyme remains in elements of the trans-Golgi reticulum. Comparative measurements of cyclic AMP in amelanotic and tyrosine-induced melanotic cells show similar basal levels. L-tyrosine and L-dopa have little or no effect, whereas MSH may cause a 1000 % peak increase in cyclic AMP levels both in amelanotic and melanotic cells. None of these agents influences cyclic GMP or inositol trisphosphate (InsP3) levels. Ln agreement with the InsP3 assays, phorbol ester (TPA) has no effect on melanization, tyrosinase activity or cell prolfleration. In conclusion, in the Bomirski amelanotic melanoma, MSH induces only partial cell differentiation associated with raised levels of cyclic AMP. Induction of melanosome synthesis and melanization by L-tyrosine or L-dopa appear to follow pathways unrelated to cyclic AMP, cyclic GMP or InsP3.
Microtubule orientation during stomatai differentiation in grasses
Authors: Cho Soon-ok , Wick Susan M.
Published: April 2021

The changing orientation of microtubules (MTs) during formation of the stomatai complex in grasses was observed by immunofluorescence microscopy, beginning with the asymmetrical division of the cell that gives rise to the guard cell mother cell, i.e. the guard cell grandmother cell. The asymmetrically placed preprophase band (PPB) of guard cell grandmother cells and hair cell mother cells is always laid down parallel to the distal end wall even when this wall is oblique to the long axis of the cell. The first step in formation of the PPB of a subsidiary cell mother cell appears to be establishment of an incomplete band of MTs. Whereas the mature PPB forms a curved line in a face view of a subsidiary cell mother cell, in this early stage MTs form fan-shaped arrays that focus on two points along the edge of the subsidiary cell mother cell. Replacement of the transversely oriented interphase microtubule band of the guard cell mother cell with the longitudinally oriented PPB involves several distinctive stages: (1) appearance of MTs directed toward the centre of the periclinal surface along the entire length of the lateral walls. (2) Appearance of another set of MTs along the entire width of both end walls, likewise focused toward the centre of the periclinal surface. Together these two groups of MTs form a cross with broadened tips in face view of the leaf. (3) Disappearance of the first set of MTs, and formation of an increasingly narrow band from the latter at the site of future cytokinesis. Although the anaphase spindles of guard cell grandmother cells, hair cell mother cells and guard cell mother cells are usually diagonally oriented relative to the site occupied previously by the PPB, the line connecting the centres of the spindle poles that are established at prophase is perpendicular to the persisting PPBs. Unlike the situation in certain other hair cells, MTs in leaf hair cells are transversely oriented even when the cells are highly elongated.
The glycoprotein of VSV accumulates in a distal Golgi compartment in the presence of CCCP
Authors: Burkhardt Janis K. , Hester Susan , Argon Yair
Published: April 2021

The post-translational modifications of the G protein of vesicular stomatitis virus, described in the preceding paper, indicate that its transport is arrested by carbonylcyanide m-chlorophenylhydra-zone (CCCP) in or near the trans-Golgi. Immuno-fluorescence microscopy of BHK-21 cells infected with vesicular stomatitis virus and treated with CCCP shows an accumulation of G protein in the Golgi area. In the same cells, the morphology of wheat germ agglutinin (WGA)-staining structures in the perinuclear region is aberrant. Using anti-BiP antibody, there is no obvious change in the structure of the endoplasmic reticulum. Electron microscopy reveals that the aberrant structures in the perinuclear region result from dilation of Golgi cistemae and accumulation of large vacuoles near the Golgi stack. The appearance of these aberrant structures is dose-dependent and they disappear after the protonophore is removed. The vast majority of the vacuoles accumulate on the trans side of the Golgi stack. A small fraction of them contain the marker enzyme thiamine pyrophosphatase (TPPase). By immunoelectron microscopy, most of the vacuoles contain G protein. We conclude that most of the Golgi-associated vacuoles are derived from a distal Golgi transport compartment, possibly the trans-Golgi reticulum, and that CCCP reversibly inhibits the transport of newly synthesized G protein through this distal compartment.
Injection of Xenopus eggs before activation, achieved by control of extracellular factors, improves plasmid dna replication after activation
Authors: Wangh Lawrence J.
Published: April 2021

Injection of molecular probes into unfertilized Xenopus eggs requires suppression of activation. But the unfertilized egg is poised for activity, and pricking, like sperm penetration, triggers the start of the first cell cycle. Methods of suppressing activation generally rely on introduction of drugs into the cell, but some of these techniques are irreversible. I report here that injection without activation can also be accomplished by simply limiting extracellular free Ca2+ to 1–2 μM. The site of injection heals, but the cortex does not contract. Gentle modification of the vitelline envelope, which causes it to become tougher, improves the rate of healing to about 100%. Healed eggs are stable for hours and can be activated when needed. Injection of a plasmid derived from type 1 bovine papilloma virus revealed that replication occurs only after activation, but preloading the DNA markedly increased the efficiency of first-round replication. DNA interaction with the unactivated egg cytoplasm may therefore be required for efficient replication of exogenous DNA. The new procedures described here are likely to be of general utility.
Nucleoside diphosphokinase, an enzyme with step changes in activity during the cell cycle of the fission yeast Schizosaccharomyces pombe II. dissociation of the steps from the DNA-division cycle after induction synchronization
Authors: Creanor J. , Mitchison J. M.
Published: April 2021

Synchrony was induced in cultures of the mitotic mutant cdc2.33 of Schizosaccharomyces pombe by shifting up an asynchronous culture to the restrictive temperature for a period of 3–5-4–5h and then shifting down to the permissive temperature. The resulting synchronous divisions had short cycle times, down to 50% of the normal cycle. The oscillatory control of nucleoside diphosphokinase activity was also synchronized by the shift-down and the activity rose in a step pattern. Unlike the situation in the normal cycle, this step pattern was dissociated from the shortened cell cycle and had a longer period and different phase relations. It may be that the normal entrainment or coupling between the cell cycle and the activity control fails if the cell cycle is too short. The period of the activity control (equal to the protein doubling time at the restrictive temperature) appears to be temperature-compensated.
Lithium ions induce prestalk-associated gene expression and inhibit prespore gene expression in Dictyostelium discoideum
Authors: Peters Dorien J. M. , Campagne Michiel M. Van Lookeren , Haastert Peter J. M. Van , Spek Wouter , Schaap Pauline
Published: April 2021

We investigated the effect of Li+ on two types of cyclic AMP-regulated gene expression and on basal and cyclic AMP-stimulated inositol 1,4,5-trisphos-phate (Ins(1,4,5)P3) levels. Li+ effectively inhibits cyclic AMP-induced prespore gene expression, half-maximal inhibition occurring at about 2mM-LiCl. In contrast, Li+ (1–3 mM) promotes the cyclic AMP-induced increase of cysteine proteinase-2 mRNA levels, and induces the expression of this prestalk-associated gene in the absence of cyclic AMP stimuli. At concentrations exceeding 4–5 mM, LiCl inhibits cysteine proteinase-2 gene expression. LiCl reduces basal Ins(1,4,5)P3 levels and decreases the cyclic AMP-induced accumulation of Ins(1,4,5)P3; both effects occur half-maximally at 2–3 mM-LiCl. These results indicate that the induction of the cysteine proteinase-2 gene by Li+ is not due to elevated levels of Ins(1,4,5)P3. It is, however, possible that inhibition of prespore gene expression by Li+ is caused by Li+-induced reduction of basal and/or stimulated Ins(1,4,5)P3 levels.
Evidence for a M
r 88 000 glycoprotein with a transmembrane association to a unique flagellum attachment region in Trypanosoma brucei
Authors: Woods Angela , Baines Anthony J. , Gull Keith
Published: April 2021

We have examined the relationship of externally accessible proteins associated with the internal cytoskeleton of procyclic Trypanosoma brucei. Two approaches were taken. First, externally disposed glycoproteins were identified with lectins and examined for their persistence and location in isolated cytoskeletons. Second, proteins containing tyrosine residues available for chemical modification on the outer surface were identified in isolated cytoskeletons and probed for glycosylation. The procyclic form of T. brucei that was employed does not express the variable surface glycoprotein. The lectin concanavalin A (ConA) bound to the outer surface of T. brucei in two discrete locations; one a narrow line close to the flagellum attachment zone on the cell body, the other at the distal tip of the flagellum itself. Of these, only the cell body labelling was detected when isolated cytoskeletons were probed with fluorescein isothiocyanate-labelled ConA. When cytoskeletons were prepared from cells labelled with gold-conjugated ConA, a narrow line of label was detected parallel to the flagellum attachment zone but was distinct from it. Only one cytoskeletal protein, of Mr 88 000, could be labelled at the cell surface by the 125l/iodogen procedure. This protein could be precipitated from SDS-solubilized cytoskeletons with ConA-agarose. These data indicate the existence of a previously undetected cytoskeletal structure, situated in the cell body, close to the point of flagellum attachment, which has a transmembrane association with an external Mr88000 glycoprotein.
Infection of algae-free Paramecium bursaria with symbiotic Chlorella sp. isolated from green paramecia: II. A timed study
Authors: Meier Renate , Wiessner Wolfgang
Published: April 2021

Algae-free Paramecium bursaria was exposed to cells of Chlorella sp. for 30 s (pulse) and then incubated in algae-free medium for periods between 0 and 15 min. During this chase the fate of the vacuoles formed during the exposure to algae was followed in order to reveal the moment of perialgal vacuole (PV) formation. PVs are characterized by always enclosing an individual algal cell and thus differ from digestive vacuoles (DVs). PVs did not appear immediately after the pulse, but were found only in cells chased for at least 3 min. In those ciliates the algae-containing vacuoles were more than 3 min old and located in the middle part of the cell. These results showed that PVs were not formed directly at the cytopharynx, but many algae were at first enclosed together in large DVs. After the release from the cytopharynx DVs undergo a sequence of fusion events during their cyclosis: fusion with acidosomes apparently occurs at the cell’s posterior end, not later than 2 min, and fusion with lysosomes in the middle region of the cell at the earliest at about 7 min, after the pinching off of a DV from the cytopharynx. Thus, PVs appeared to develop from condensing DVs after acidosomal but before lysosomal fusion. As the first step, part of the DV enclosing an individual algal cell must detach from the large vacuole. Further steps and the implications of the mechanism of PV formation resulting in the re-establishment of endosymbionts in P. bursaria are discussed.
A comparison of the biological activities of the cell-adhesive proteins vitronectin and fibronectin
Authors: Underwood P. A. , Bennett F. A.
Published: April 2021

The effects of vitronectin and fibronectin upon the attachment and growth of bovine corneal endothelial cells (BCE) and BHK-21 cells were compared. Similar dose-response curves for cell attachment to the substratum were obtained for both molecules and both cell types, although BCE cells exhibited a slight preference for vitronectin, and BHK cells for fibronectin. When, however, cells were plated in medium containing bovine serum stripped of fibronectin, they attached and grew normally, whereas in medium containing serum stripped of vitronectin, cells either failed to attach (BHK-21) or attached but exhibited poor cell spreading and growth. This dependence of cells upon vitronectin, rather than fibronectin, in serum for cell attachment, was shown to be due to a failure of fibronectin to coat the substratum in the presence of other serum proteins. Vitronectin was able to coat the substratum efficiently in the presence of other serum proteins. Although dependent upon vitronectin for adhesion to the substratum, bovine endothelial cells were unable to synthesize endogenous vitronectin.
Ultrastructure of Fanconi anemia fibroblasts
Authors: Willingale-Theune J. , Schweiger M. , Hirsch-Kauffmann M. , Meek A. E. , Paulin-Levasseur M. , Traub P.
Published: April 2021

Employing indirect immunofluorescence and conventional electron microscopy, gross nuclear aberrations were observed in cultured interphase fibroblasts derived from a patient suffering from Fanconi’s anemia (FA). Such aberrations were predominantly expressed in cells at high passages between 28 and 34. The structure of the nuclei appeared compound in nature, often consisting of two to three nuclear fragments connected to each other by thin nuclear bridges containing chromatin and nuclear lamin material. In other cases, the nuclei appeared lobed or budded but the cells did not contain distinct nuclear fragments. Chromatin was conspicuously absent from some nuclear lobes, revealing empty, cage-like structures comprising nuclear lamin material. Micronuclei were often abundant in the perinuclear cytoplasm but in some instances they appeared to be composed of chromatin lacking a delineating nuclear lamin matrix. Residual cytoskeletons examined by whole-mount electron microscopy revealed a network of inter mediate filaments (IFs) within FA fibroblasts forming a bridge between the plasma membrane and the nucleus or its major fragments. In addition, there were thinner, 3–4 nm filaments connecting individual IFs with the surface of the nucleus. Micronuclei that were not connected to the main nuclear body, but which were delineated by a distinct lamina and possessed nuclear pores, did not appear to be anchored to the IF network. Multinuclearity, nuclear fragmentation, irregular chromatin distribution and inter-nuclear chromatin/lamin bridges might result from a failure in the redistribution of chromatin to sister nuclei, incomplete cytokinesis and proliferation of nuclear envelope material. These phenomena point to precocious aging of FA fibroblasts and may occur as a consequence of spontaneous damage to the sister chromatids or through the action of DNA-toxic agents.
Cellular localization of the serh surface antigen in Tetrahymena thermophila
Authors: Bolivar Ignacio , Guiard-Maffia Jacqueline
Published: April 2021

We have purified the SerH surface antigen of T. thermophila by a novel and simple procedure and produced specific antisera against it. By the use of various immunochemical techniques, we have investigated the intracellular distribution of the antigen and shown that SerH is not only abundant in the cortex but also in the digestive apparatus of the ciliate. In each of these two localizations, SerH occupies a variety of compartments: in the cortex it can be found in the surface coat, the exocytotic mucocysts, the endocytotic parasomal sacs and as an integral protein of the membrane; in the digestive apparatus, SerH is found around ingested bacteria, in the cytoplasm surrounding the cytopharynx and the forming food vacuoles, and, seemingly, as a membrane-associated protein in young food vacuoles. Pulse-chase experiments have shown that feeding dramatically increases the global turnover of SerH. Besides these localizations, SerH is principally found in clumps around dense intracytoplasmic spheres, which could be mucocyst precursors. Indirect evidence is presented that SerH is routed to the peripheral or extracellular compartments via the mucocysts. The antigen is absent from alveolar, nuclear or mitochondrial membranes. We propose that SerH covers any membrane in contact or future contact with the extracellular medium.
Induction of repressible acid phosphatase by unsaturated fatty acid in Saccharomyces cerevisiae
Authors: Doi Syuichi , Watanabe Masayasu , Tanabe Kazuyuki , Nakasako Michiko , Yoshimura Masao
Published: April 2021

We studied the induction of acid phosphatase (APase) by fatty acids in Saccharomyces cerevisiae. S. cerevisiae has two types of APase: constitutive and repressible enzymes. The synthesis of the latter APase is normally derepressed by depletion of inorganic phosphate (P1) in the incubation medium. Of the saturated and unsaturated fatty acids tested, linoleic, linolenic and arachidonic acids induced the synthesis of APase even in the presence of a high concentration of P1, whereas palmitic, stearic and oleic acids did not. De novo protein synthesis but not stimulation of secretion of the enzyme was required for the induction. Genetic analyses using plasmids carrying the genes, PHO5 and PHO3, that code for repressible APase and constitutive APase, respectively, showed that linolenic acid induced the formation of repressible APase. Linolenic acid inhibited the uptake of exogenous 32P1 and simultaneously lowered the intracellular level of P1. These circumstances indicate that linolenic acid-induced derepression of respressible APase is primarily caused by a fall in the intracellular level of P1. However, cells that had been preincubated in the presence of a high concentration of P1 produced APase shortly after the addition of linolenic acid. It is, therefore, suggested that, as well as a normal regulatory mechanism for derepression of repressible APase, a mechanism independent of the external level of P1 participates in the induction of repressible APase by linolenic acid.
Bradykinin receptor number and sensitivity to ligand stimulation of mitogenesis is increased by expression of a mutant ras oncogene
Authors: Roberts R.A. , Gullick W.J.
Published: April 2021

Bradykinin is a nine amino acid peptide of the kinin family believed to play a role in pain mediation and in the regulation of blood pressure, fluid balance and smooth muscle contraction. Here we demonstrate that bradykinin is also a potent mitogen for a mutant Ha-ras-transfected cell line, Rat 13. The Rat 13 cells display two binding sites for bradykinin: a moderate number (52,000) of high affinity sites (Kd = 4.9 nM) co-exist with a much smaller number (1100) of very high-affinity sites (Kd = 2.7 pM). Ligand binding stimulates mitogenesis through the lower affinity receptors, which are classified as B2. These receptors are down-regulated in response to ligand. In contrast, Rat 1 cells (the cell line from which Rat 13 was derived) have only 4000 receptors per cell in total and respond weakly to bradykinin as a mitogen. Thus, expression of a mutant ras protein in Rat 13 cells increases their expression of the bradykinin receptor and their sensitivity to ligand stimulation of mitogenesis. Additional binding studies demonstrate that human A431 epithelial cells and Swiss mouse 3T3 fibroblasts also possess high-affinity sites for bradykinin.
Retinoic acid-induced inhibition of lung colonization and changes in the synthesis and properties of glycosaminoglycans of metastatic b16 melanoma cells
Authors: Edward Michael , MacKie Rona M.
Published: April 2021

The effect of all-trans retinoic acid on metastatic B16 melanoma lung colonization and synthesis and properties of glycosaminoglycans was examined. Injection of tumour cells, pretreated with 10−6M-retinoic acid or grown to low density, into the tail vein of syngeneic C57 mice produced significantly fewer pulmonary tumours compared to subconfluent control cells. By cochromatography of glycos-aminoglycans isolated from control ([14C]glucosamine-labelled) and 10−6M-retinoic acid-treated ([3H]glucosamine-labelled) cells on DEAE ion-exchange columns, differences in elution profiles were detected. Chondroitin sulphates isolated from retinoic acid-treated cells eluted at a lower salt concentration than those from control cells, while retinoic acid-treated cells synthesised heparan sulphates of a higher charge density than heparans from control cultures. These changes were apparent in both medium and trypsin-releasable fractions. Retinoic acid-treated cultures were seeded so that they were of a similar density to control cultures when harvested, as cell density was shown to affect glycosaminoglycan synthesis, the glycosaminoglycans from low-density cultures having similar properties to those isolated from retinoic acid-treated cultures. Retinoic acid treatment also reduced the overall synthesis of glycosaminoglycans while having little effect on the composition or distribution between medium, trypsin-releasable and cell-associated fractions.

These observed changes in glycosaminoglycans may, in part, be responsible for retinoic acid-induced inhibition of lung colonization, and reduced adhesion to basement membrane components, which we have previously demonstrated.
Soluble proteins as modulators of the exocytotic reaction of permeabilised rat mast cells
Authors: Koffer A. , Gomperts B. D.
Published: April 2021

This study addresses the question of the role of cytoplasmic proteins in exocytosis from permeabilised rat mast cells. We have used two different methods of cell permeabilisation (ATP4− and streptolysin O) to regulate the size of the plasma membrane lesions, and thus to dictate the rate and extent of efflux of the cytosolic proteins, and compared the secretory response of the two preparations. We report evidence for the existence of two factors present in the cytosol, which affect the exocytotic mechanism in opposing manners. One of these is required for the maintenance of cell responsiveness; it is retained for more than 120 min by ATP4−-permeabilised cells but lost within 60 min from cells permeabilised by streptolysin O. The other factor, which leaks immediately from cells treated from streptolysin O, but only gradually from cells treated with ATP4−, has the effect of suppressing the affinity for both Ca2+ and guanine nucleotide in the exocytotic reaction.
Sequestrocytes: a manifestation of transcellular cross-bonding of the red cell membrane in sickle cell anemia
Authors: Weinstein Ronald S. , Warth James A. , Near Karen , Marikovsky Yehuda
Published: April 2021

An unusual form of red cells (called sequestrocytes) that circulate in the peripheral blood of patients with sickle cell anemia has been identified. In wet-mount light microscopy preparations, sequestro-cytes appear as massively vacuolated erythrocytes. We have shown by transmission electron microscopy and scanning electron microscopy that the morphology of sequestrocytes can be accounted for on the basis of transcellular cross-bonding of the cell membrane. Initially, endocytotic vesicles span the cytoplasmic space, forming putative cytoskeletal fusion zones. Points of fusion of the membrane skeleton bridging the cytoplasmic compartment expand laterally to form linear fusion zones that entrap lakes of hemoglobin within pseudo vacuoles.

Sequestrocytes can be isolated in the densest layer (1.159 g ml−1 or greater) of an arabinogalactan density gradient. These cells can be generated in increased numbers in sickle cell blood by incubating samples in 1.5 mM-acetylphenylhydrazine (APH) solution for two hours at 37°C. Their formation is partially blocked by incubation with the reducing agent, dithiothreitol (DTT). Our results suggest that these cells represent an expression of oxidative membrane injury in sickle cell anemia.
Confocal fluorescence microscopy with the tandem scanning light microscope
Authors: Wright Shirley J. , Walker James S. , Schatten Heide , Simerly Calvin , Mccarthy Jon J. , Schatten Gerald
Published: April 2021

Applications of the tandem scanning confocal microscope (TSM) to fluorescence microscopy and its ability to resolve fluorescent biological structures are described. The TSM, in conjunction with a cooled charge-coupled device (cooled CCD) and conventional epifluorescence light source and filter sets, provided high-resolution, confocal data, so that different fluorescent cellular components were distinguished in three dimensions within the same cell. One of the unique features of the TSM is the ability to image fluorochromes excited by ultraviolet light (e.g. Hoechst, DAPI) in addition to fluorescein and rhodamine. Since the illuminationis dim, photobleaching is insignificant and prolonged viewing of living specimens is possible. Series of optical sections taken in the Z-axis with the TSM were reproduced as stereo images and threedimensional reconstructions. These data show that the TSM is potentially a powerful tool in fluorescence microscopy for determining three-dimensional relationships of complex structures within cells labeled with multiple fluorochromes.
Actin is associated with the formation of the cell wall in reverting protoplasts of the fission yeast Schizosaccharomyces pombe
Authors: Kobori Hiromi , Yamada Naoko , Taki Akiko , Osumi Masako
Published: April 2021

To clarify the involvement of actin in the formation of the yeast cell wall, reverting protoplasts of Schizosaccharomyces pombe were used as a simple model system. Actin of reverting protoplasts was labeled with rhodamine-conjugated phalloidin and observed by conventional fluorescence microscopy and laser scanning confocal microscopy. A close spatial as well as temporal relationship between actin and cell wall formation was observed in protoplast reversion. That is, the site of actin‘dots’ in the reverting protoplasts coincided with the site of new wall formation and the timing of rearrangement of actin coincided with the initiation of cell wall formation and with the timing of cell wall expansion.

Treatment of reverting protoplasts with cytochalasin D (CD) further clarified the close relationship between actin and cell wall organization. The effect of CD was dose dependent. A high dose of CD caused the absence of actin as well as the complete inhibition of cell wall formation. A low dose of CD caused weakly stained unlocalized actin, which induced grossly aberrant cell wall deposition as well as substantial changes in the morphology of the reverting protoplasts. These results demonstrated that actin is associated with initiation of cell wall formation, the proper deposition of cell wall materials, and maintaining the normal morphology of reverting protoplasts. Scanning electron microscopy revealed the presence of a fibrillar net structure on the surface of non-treated control reverting protoplasts. However, the absence of a fibrillar network on the surface of reverting protoplasts was observed in the presence of a high concentration of CD. Lack of localization of microfibrils as well as poor development of the fibrillar network were also observed in the presence of a low concentration of CD. Recovery experiments confirmed the close relationship between actin and cell wall formation.