Ultrastructural differences between wall apices of growing and non-growing hyphae ofSchizophyllum commune

Summary Newly synthesized chitin at the hyphal apex ofSchizophyllum commune was shown to be highly susceptible to chitinase degradation and solubilization by dilute mineral acid. With time this chitin became gradually more resistant to these treatments. With a combination of the shadow-cast technique and electron microscopic autoradiography it could be shown that this process occurred as the newly synthesized chitin moved into subapical parts of growing hyphae but also in non-growing apices which had ceased growth after incorporation of theN-acetyl[6-³H]glucosamine. These results are in agreement with a model which explains apical morphogenesis by assuming that the newly synthesized wall material at the apex is plastic due to the presence of individual polymer chains but becomes rigidified because of subsequent physical and chemical changes involving these polymers.Dedicated to Dr. A.Quispel, Professor of Botany at the University of Leiden, on occasion of his retirement.     

Autoradiographic study of hyphal cell wall synthesis of Saprolegnia

Incorporation of radioactive glucose in growing apices of Saprolegnia monoïca hyphae were examined by electron microscopic autoradiography. ³H glucose labelling indicates that dictyosomes and apical vesicles do not contain much polysaccharide and that glucan synthesis occurs at the cell surface. ¹⁴C glucose labelling shows that incorporation was chased from the cell walls during hyphal morphogenesis.     

Appearance and distribution of carbohydrate-rich macromolecules on the epithelial surface of the developing rat palatal shelf.

The synthesis and appearance of carbohydrate-rich macromolecules by epithelial cells of the developing secondary palate was examined with concanavalin A (CON A) binding and [³H]glucosamine labeling. The amount of [¹²⁵I]CON A bound to the epithelial surface of the rat palatal shelf in vitro increased from day 15 of gestation to day 16 when initial adhesion to the opposite shelf occurs in vivo. Visualization of CON A binding by electron microscopy using the peroxidase method revealed a dramatic increase in binding between days 15 and 16 of gestation, most apparent on the medial-edge epithelial surface. The incorporation in vivo of [³H]glucosamine during this period into the medial-edge epithelial cells was detected with autoradiography. These results show that a glycoprotein-rich surface material appears on the superficial cells of the medial-edge epithelium prior to adhesion of the apposing shelves.     

Biological evaluation of a series of 2-acetamido-2-deoxy-D-glucose analogs towards cellular glycosaminoglycan and protein synthesis in vitro

Using primary hepatocytes in culture, various 2-acetamido-2-deoxy-D-glucose (GlcNAc) analogs were examined for their effects on the incorporation of D-[³H]glucosamine, [³⁵S]sulfate, and L-[¹⁴C]leucine into cellular glycoconjugates. A series of acetylated GlcNAc analogs, namely methyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-(3) and β-D-glucopyranoside (4) and 2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-D-glucopyranose (5), exhibited a concentration-dependent reduction of D-[³H]glucosamine, but not of [³⁵S]sulfate incorporation into isolated glycosaminoglycans (GAGs), without affecting L-[¹⁴C]leucine incorporation into total protein synthesis. These results suggest that analogs 3–5 exhibit an inhibitory effect on D-[³H]glucosamine incorporation into isolated GAGs by diluting the specific activity of cellular D-[³H]glucosamine and by competing for the same metabolic pathways. In the case of the corresponding series of 4-deoxy-GlcNAc analogs, namely methyl 2-acetamido-3,6-di-O-acetyl-2,4-dideoxy-α-(6) and β-D-xylo-hexopyranoside (7) and 2-acetamido-1,3,6-tri-O-acetyl-2,4-dideoxy-D-xylo-hexopyranose (8), compound 8 at 1.0 mM exhibited the greatest reduction of D-[³H]glucosamine and [³⁵S]sulfate incorporation into isolated GAGs, namely to ∼7% of controls, and a moderate inhibition of total protein synthesis, namely to 60% of controls. Exogenous uridine was able to restore the inhibition of total protein synthesis by compound 8 at 1.0 mM. Isolated GAGs from cultures treated with compound 8 were shown to be smaller in size (∼40 kDa) than for control cultures (∼77 kDa). These results suggest that the inhibitory effects of compound 8 on cellular GAG synthesis may be mediated by the incorporation of a 4-deoxy moiety into GAGs resulting in premature chain termination and/or by its serving as an enzymatic inhibitor of the normal sugar metabolites. The inhibition of total protein synthesis from cultures treated with compound 8 suggests a uridine trapping mechanism which would result in the depletion of UTP pools and cause the inhibition of total protein synthesis. A 1-deoxy-GlcNAc analog, namely 2-acetamido-3,4,6-tri-O-acetyl-1,5-anhydro-2-deoxy-D-glucitol (9), also exhibited a reduction in both D -[³H]glucosamine and [³⁵S]sulfate incorporation into isolated GAGs by 19 and 57%, of the control cells, respectively, at 1.0 mM without affecting total protein synthesis. The inability of compound 9 to form a UDP-sugar and, hence, be incorporated into GAGs presents another metabolic route for the inhibition of cellular GAG synthesis. Potential metabolic routes for each analog's effects are presented.     

Primary cultures of heart cells from the scallp Pecten maximus (mollusca-bivalvia)

Summary Primary cultures of Pecten maximus heart cells, isolated by an enzymatic procedure, were routinely obtained with a high level of reproducibility in a simple medium based on sterile seawater. Cells attached to the plastic substratum without the need to add a special factor. The number of adhering cells gradually increased with the time of culture. Two types of adhering cells were observed: epitheliallike cells and fibroblastlike cells, which were more numerous. The latter cells were identified as myocytes by electron microscopy and immunofluorescent staining. Results obtained by autoradiography, after incorporation of [¹⁴C]leucine, [³H]thymidine, and [¹⁴C]acetate, confirmed functional activity of the cells. These cultures were maintained viable in vitro during at least 1 mo.     

Metabolic coupling between cultured pancreatic B-cells

The transfer of [³H]uridine nucleotides from donor (uridine-loaded) to recipient (thymidine-prelabelled) pancreatic endocrine islet cells in monolayer culture was qualitatively and quantitatively assessed by light and electron microscope autoradiography. Recipient cells showed a labelled cytoplasm only when they were in contact with donor cells or positive recipients. Controls indicated that the labelling was not due to the incorporation of [³H]uridine, ³H nucleotides or nucleic acids lost by donor cells in the medium. Quantitation showed that cytoplasmic labelling of positive recipient cells was higher than cellular background, but lower than the cytoplasmic labelling of donor cells. These data indicate that label in recipient cells was derived from donors by direct intercellular transfer of ³H nucleotides. Differentiated insulin-producing cells (B cells) were involved in the exchange.     

Thyroxine-stimulated synthesis of microvillus membrane glycoproteins during culture of chick embryonic duodenum

The effect of thyroxine on biosynthesis of microvillus membrane glycoproteins has been investigated in organ culture of 18-day-old chick embryonic duodenum. Explants incorporate [³H]leucine and [³H]glucosamine continuously, and overall incorporation is enhanced by 10 nM thyroxine during 48 h of labeling; this increase in radioactivity is associated with vesicles released from the microvilli. Light microscope autoradiography, pulse labeling of brush border fragments, and pulse chase experiments reveal that [³H]glucosamine is incorporated into brush border at an increasing rate during culture, and that newly synthesized glycoproteins are discharged into the medium along with brush border enzymes (alkaline phosphatase and maltase). These results suggest that thyroxine stimulates biosynthesis of microvillus membrane glycoproteins, in addition to stimulating vesiculation of the membrane. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of ³H-labeled vesicles and brush border fragments show that [³H]leucine and [³H]glucosamine are incorporated into proteins of high molecular weight. Two protein bands are identified as alkaline phosphatase and maltase. Thyroxine stimulates glycosylation of these enzymes, but does not change protein patterns. Radioactivity assay of alkaline phosphatase- and maltase-active gel slices suggests that thyroxine stimulation of these enzyme activities during culture is not correlated with de novo synthesis of these proteins.     

Evidence for synthesis in vitro of neutral lipids by isolated oocytes of Perinereis cultrifera (Annelida,Polychaeta). A biochemical and autoradiographic study

Summary

Isolated oocytes of Perinereis cultrifera have been incubated in culture media with added [³H]glycerol, [¹⁴C]butyric acid or [¹⁴C]oleic acid. The principal neutral lipid synthesized was triacylglycerol, although incorporation of radioactivity into other lipid categories (sterol, fatty acid, wax ester) was also observed. A more significant percentage of triacylglycerol was labelled after incubation with [³H]glycerol and [¹⁴C]oleic acid than with [¹⁴C]butyric acid. With this precursor, monoacylglycerol appears to be the class of lipid compartment which initially show the most radioactivity. Electron microscopic autoradiography has revealed that labelling after incorporation of glycerol was mainly localized on the lipid droplets but not on the yolk granules. A second metabolic pathway is represented by phospholipid membrane synthesis.     

A STUDY OF THE ULTRASTRUCTURAL LOCALIZATION OF HAIR KERATIN SYNTHESIS UTILIZING ELECTRON MICROSCOPIC AUTORADIOGRAPHY IN A MAGNETIC FIELD

The sites of the incorporation of labeled cystine into keratinizing structures were studied in electron microscopic autoradiographs. The tracer used was cystine labeled with S³⁵ emitting long-range ionizing particles. During exposure for 1 to 2 months, according to our method of electron microscopic autoradiography, emulsion-coated specimens were exposed to a static magnetic field which appeared to result in a marked increase in the number of reacted silver grains. In young Swiss mice receiving intraperitoneal injections at 1, 3, and 6 hours before biopsy, conventional autoradiography demonstrated that S³⁵-cystine was intensely localized in the keratogenous zone of anagen hair follicles, and that the radioactivity there increased in intensity progressively with time while the radioactivity in the hair bulb always remained very low. Our observations with electron microscopic autoradiography in a magnetic field appeared to indicate that at 3 and 6 hours after injection the S³⁵-cystine was directly and specifically incorporated into tonofibrils in the hair cortex and into amorphous keratin granules of the hair cuticle layer, possibly without any particular concentration of this substance in the other cellular components. There seemed to be an appreciable concentration of cystine in tonofibrils of the cuticle of the inner root sheath. However, trichohyalin granules in the hair medulla and inner root sheath failed to show any evidence of cystine concentration. The improved sensitivity of the electron microscopic autoradiography with S³⁵-cystine appeared to be partly due to the application of a static magnetic field. However, the reason for this could not be explained theoretically.     

Correlation of cell division and specific protein production during the course of lymphoid cell differentiation

Mice were immunized with horseradish peroxidase and injected with [³H] thymidine. Popliteal lymph node cells were submitted to electron microscopic immunocytochemistry and high resolution autoradiography in order to correlate antibody production and ability to undergo cell division at various stages of lymphoid cell differentiation. Antibody synthesis started in blast cells and increased steadily until the mature plasma cell stage was reached. Thymidine incorporation was highest in blastoid cells and decreased continuously afterwards. Chromatin dispersion was found to be paralleled by thymidine incorporation. This observation and data of other authors seem to indicate that chromatin dispersion is a prerequisite for replication. Almost all mature plasma cells were devoid of thymidine incorporation. This confirms that they are end cells apparently unable to divide.     

Cell wall formation in yeast

Summary Incorporation of tritiated glucose into cell walls of growingSaccharomyces cerevisiac andSchizosaccharomyces pombe was studied using electron microscopic autoradiography. The pattern and the extent of labelling ofS. cerevisiae cell walls depended on the cell stage in the cell cycle. Quantitative evaluation of autoradiographs showed that the highest rate of wall synthesis took place during bud growth. The incorporation of new material into the wall of growing bud showed an increasing rate with the magnitude of the bud. The incorporation into the mother cell wall was almost negligible during bud growth. The rate of wall synthesis in double cells decreased during cell division. This period and that before new bud initiation was found to be the time of substantially reduced rate of wall replication inS. cerevisiae. A significant random incorporation was observed into the walls of post-division adult cells, both parental and daughter. The cell walls ofS. pombe were labelled almost exclusively at growing tips. The incorporation of tritiated carbohydrates into non-extensile regions ofS. pombe cell walls was found to be only about 5% of the total wall labelling.     

Purification and characterization of bacteriophage receptor on Veillonella rodentium cells

Veillonellophage N2 adsorbed to polysaccharides (PSs) on Veillonella rodentium ATCC 17743 cell wall, and the bacteriophage receptor contained only glucosamine. D(+)-glucosamine hydrochloride (Sigma) also adsorbed the veillonellophage N2. These results therefore indicate that the receptor to the veillonellophage N2 is cell wall PSs. The PSs of the host cells as receptor have been characterized. Glucosamine accounted for approximately 100% of the weight of the PSs. The PSs which were partially resolved by Sephadex G-75 chromatography comprised approximately four glucosamine units. Their primary structure was determined by 400 MHz n.m.r. spectroscopy. One- and two-dimensional ¹H-nmr experiments showed the PS to be a branched polymer. Glucosamine linkage was detected in one of the branches.     

Low concentrations of dimethyl sulphoxide accelerate conjugation and incorporation of glycine and glucosamine intetrahymena

Summary Dimethyl sulphoxide at relatively low comentrations, 0.01 to 1 mM, enhanced the conjugation and cell-to-cell adhesion of complementary strains of matingTetrahymena thermophila. The time required to form stable conjugates was reduced by dimethyl sulphoxide. This chemical stimulated the uptake of glycine and glucosamine from the suspending media. Incorporation of 2-¹⁴C-glycine and 6-³H-D-glucosamine into protein and glycoprotein was enhanced in whole cells, surface membrane and cilia. Incorporation of glucosamine into the microsomal fraction was increased in the dimethyl sulphoxidetreated cells while there was little change in glycine incorporation. There were no detectable changes in glycine and glucosamine incorporation into the nuclear fractions isolated from conjugatingTetrahymena exposed to dimethyl sulphoxide.     

Ultrastructure and autoradiography of dormant and activated parenchyma ofHelianthus tuberosus

Summary Parenchyma cells of dormant tubers ofHelianthus tuberosus L. cv. OB1 (Jerusalem artichoke) contain a very low amount of hormones, therefore they respond to 2,4-D or IAA treatment by dividing and synthesizing RNA, DNA, and polyamines.In particular the activation of the dormant tissues induces an early synthesis of DNA, which reaches the maximum at 3 hours, much before the beginning of the S phase (12 hours). By supplying [6-³H] thymidine and carrying out electron microscopic autoradiography, we were able to determine that plastids and mitochondria were the organelles responsible for this early synthesis while the DNA in the nucleus first appeared labeled at 15 hours.In addition, ultrastructural observations carried out to compare the dormant cells with activated ones, showed an increase in the nucleolar volume, a different organization of the tubular complex of the plastids and several other ultrastructural changes which indicate that at 3 hours some fundamental metabolic processes are already active; they become even more evident later on.The implications of these results in the physiology of the tuber cells during activation are discussed.     

Biosynthesis of cell-wall polysaccharides in cultured carrot cells

Biosynthesis of the cell wall in carrot cells (Daucus carota L.) cultured in a synthetic liquid medium was studied by measuring the incorporation of radioactive glucose and myo-inositol (MI). When the cells were fed with [¹⁴C]glucose in the presence of 0.01% MI, the label soon appeared in the neutral sugars in the cell wall but little radioactivity was found in the uronic-acid residues even after a prolonged incubation. On the other hand, radioactivity derived from [³H]MI was found to be distributed among uronic acids and pentoses but not in the hexose residues in the wall. The data indicate that MI is an important intermediate for the synthesis of acidic sugars in the wall of cultured carrot cells.Abbreviation MI myo-inositol     

Differential patterns of mitochondrial,chloroplastic and nuclear DNA synthesis in the synchronous cell cycle of Chlamydomonas reinhardtii

Nuclear DNA (ncDNA) synthesis in Chlamydomonas reinhardtii was measured by both ³²P[or-thophosphoric acid] (³²P) and [¹⁴C]adenine incorporation and found to be highly synchronous. Ca. 85% of incorporation was confined to the first 6 h of the dark period of a synchronized regime consisting of an alternating light-dark period of 12 h each. In contrast, no such synchronous incorporation pattern was found for chloroplast (cp) and mitochondrial (mt) DNAs in the same cell population. These two organellar DNAs also exhibited different ³²P-incorporation patterns in the cell cycle. Considerable amounts of ³²P were incorporated into cpDNA throughout the light-dark synchronous cycle under both mixo- and phototrophic growth conditions, although the second 6-h light period under phototrophy showed an increase not apparent under mixotrophy. This change in growth conditions did not affect ³²P incorporation into mtDNA, which was found throughout the cell cycle, with a modest peak in the first 6-h of the dark period. The pattern of [³H]thymidine incorporation into cpDNA was also determined. Under synchronous phototrophic conditions, this pattern was quite different from that obtained with ³²P. Most [³H]thymidine incorporation occurred during the light period of the synchronous cycle; this period had been shown previously by density transfer experiments to be the time of cpDNA duplication. Such preferential [³H]thymidine incorporation into cpDNA in the light period was not observed under mixotrophic synchronous growth conditions; in these, [³H]thymidine incorporation was detected throughout the cell cycle. This lack of coincidence between the patterns of ³²P- and of [³H]thymidine incorporation into cpDNA during the synchronous cell cycle indicates that in addition to replication, the considerably reiterated organelle-DNA molecules may also regularly undergo an extensive repair process during each cell cycle.     

Effect of tunicamycin on epidermal glycoprotein and glycosaminoglycan synthesis in vitro

1. When pig ear skin slices were cultured for 18h in the presence of 1μg of tunicamycin/ml the incorporation of d-[³H]glucosamine into the epidermis, solubilized with 8m-urea/5% (w/v) sodium dodecyl sulphate, was inhibited by 45–55%. This degree of inhibition was not increased by using up to 5μg of tunicamycin/ml or by treating the skin slices with tunicamycin for up to 8 days. The incorporation of (U-¹⁴C)-labelled l-amino acids under these conditions was not affected by tunicamycin. Polyacrylamide-gel electrophoresis indicated that the labelling of the major glycosaminoglycan peak with d-[³H]glucosamine was unaffected, whereas that of the faster migrating glycoprotein components was considerably decreased in the presence of tunicamycin. 2. Subcellular fractionation indicated that tunicamycin specifically inhibited the incorporation of d-[³H]glucosamine but not of (U-¹⁴C)-labelled l-amino acids into particulate (mainly plasma-membrane) glycoproteins by about 70%. The labelling of soluble glycoproteins was hardly affected. Polyacrylamide-gel electrophoresis of the plasma-membrane fraction showed decreased d-[³H]glucosamine incorporation into all glycoprotein components, indicating that the plasma-membrane glycoproteins contained mainly N-asparagine-linked oligosaccharides. 3. Cellulose acetate electrophoresis of both cellular and extracellular glycosaminoglycans showed that tunicamycin had no significant effect on the synthesis of the major component, hyaluronic acid. However, the incorporation of both d-[³H]glucosamine and ³⁵SO₄²⁻ into sulphated glycosaminoglycans was inhibited by about 50%. This inhibition was partially overcome, at least in the cellular fraction, by 2mm-p-nitrophenyl β-d-xyloside indicating that tunicamycin-treated epidermis retained the ability to synthesize sulphated glycosaminoglycan chains. Tunicamycin may affect the synthesis and/or degradation of proteoglycan core proteins or the xylosyltransferase. 4. Electron-microscopic examination of epidermis treated with tunicamycin for up to 4 days revealed no significant changes in cell-surface morphology or in epidermal-cell adhesion. Either N-asparagine-linked carbohydrates play little role in epidermal-cell adhesion or more probably there is little turnover of these components in epidermal adhesive structures such as desmosomes and hemidesmosomes during organ culture.     

An autoradiographic study of RNA synthesis during maturation and germination of pollen grains ofHyoscyamus niger

Summary The pattern of RNA synthesis during maturation and germination of pollen grains ofHyoscyamus niger was studied using³H-uridine autoradiography. Incorporation of label during pollen maturation was periodic with peak RNA synthesis occurring in the uninucleate, nonvacuolate pollen grains and in the vegetative cell of the bicellular pollen grains. During the early stages of germination, isotope incorporation occurred predominantly in the nucleus of the vegetative cell with little or no incorporation in the generative cell. With the appearance of the pollen tube, incorporation of³H-uridine in the vegetative cell nucleus decreased and completely disappeared at later stages of germination. No incorporation of isotope was observed in the sperms formed in the pollen tube by the division of the generative cell. From a comparison of the results of this study with those of previous works on RNA synthesis during pollen embryogenesis in cultured anthers ofH. niger, it is concluded that in contrast to embryogenic development, there is no requirement for sustained RNA synthesis by the generative cell nucleus for normal gametophytic development.     

Zero order kinetics of cell wall turnover inStaphylococcus aureus

Cells exponentially grown from four strains ofS. aureus (SG 511, H, 52A5G, and248 PN-1) and uniformly labeled in their walls with³H-N-acetylglucosamine, were found to turn over their old walls at constant rates of up to 25% per generation. Wall turnover was not observed to follow first order kinetics, thus ruling out the implication that maintenance of normal wall thickness was achieved by a random distribution of new wall components in the old wall. Instead, wall turnover in all cases strictly followed zero order kinetics, indicating that newly synthesized wall material was placed layer by layer beneath the inner surface of the old cell wall. This finding correlates with evidence obtained from earlier electron microscopic investigations into the regeneration of the staphylococcal cell wall after chloramphenicol treatment. Based on the experimental data presented, a simplified model for wall turnover of the growing staphylococcal cell was proposed. The model also takes into account the finding, derived from additional experiments with strainSG 511, that the total cell wall turned over at a somewhat higher rate than the old portions of the wall. The rates of cell wall turnover found inS. aureus SG 511 are the highest reported to date for pathogenic bacteria. The medical implications of this finding were discussed.     

A technique for the electron microscopic autoradiography of Al adenosine receptors in brain tissue

Summary A procedure for the electron microscopic autoradiography of Al adenosine receptors is described. Fresh tissue slices from rat hippocampus were incubated with the radioactive adenosine analogs: Cyclohexyl[³H]adenosine, 5-N-ethylcarboxamido[³H]adenosine or [¹²⁵I]-iodohydroxyphenylisopropyladenosine. Various fixation agents were tested with respect to the retention of these ligands by the tissue. While most of the ligands were lost in aldehyde fixation they were retained by osmium tetroxide probably via a crosslinking reaction. The final method of choice was an aldehyde prefixation (in the case of [¹²⁵I]-iodohydroxyphenylisopropyladenosine with 4% buffered paraformaldehyde) during which more than 90% of the nonspecifically bound ligands were washed out while 40% of the specifically bound ligands remained. Subsequent fixation with osmium tetroxide (1%) allowed a standard protocoll for dehydration and embedding to be used with only minimal (less than 5%) further loss of the ligands. Electron microscopic autoradiography provided evidence for a specific distribution of the binding sites for [¹²⁵I]-iodohydroxyphenylisopropyladenosine.Abbreviations GA Glutardialdehyde - PFA Paraformaldehyde - OsO₄ Osmiumtetroxide - CHA Cyclohexyladenosine - NECA N-ethyl-carboxamidoadenosine - PIA Phenylisopropyladenosine - I-HPIA Iodohydroxyphenylisopropyladenosine - HPIA Hydroxyphenylisopropyladenosine