Induction of the Synthesis of Melanin and Pteridine in Cells Isolated from the Axolotl Embryo

It has previously been reported that when LiCl and tyrosine is added to ectodermal cells isolated from the blastula of Ambystoma mexicanum , then the synthesis of melanin is initiated in cells not normally engaged in this activity (mesenchyme cells, nerve cells and undifferentiated animal cells). In the present paper it has been shown that to obtain this effect tyrosine (0.02 mM) has to be present in the culture medium during at least one of the first seven days of culture, thus several days before melanin is produced. It is concluded that the added tyrosine is acting as an inductor of, and not as a substrate for the synthesis of melanin.
In the normal cultures it is possible to observe the spontaneous formation of yellow cells, indicating that they have produced pteridine. These cells are spherical, suggesting that they are undifferentiated embryonic cells. GTP is a precursor in the synthesis of pteridine, and in analogy with the observations made with tyrosine it was found that in the presence of LiCl a number of different cell types elaborate pteridine when GTP (0.1 mM) is added to the medium. Also in this case was it possible to show that GTP acts as an inductor, not as a substrate.     

Induction of precocious melanogenesis of pigment cells in cultures of neuroretinal cells of chick embryo by amphotericin B

Summary When dissociated neuroretinal cells of the 9-day-old chick embryo were cultured, the cells formed monolayer sheets of somewhat flattened epithelial cells within 15 days after inoculation. During 15 to 30 days, numerous foci of non-pigmented epithelial cells were formed. During 30 to 50 days, melanin appeared in the cells of these foci. When amphotericin B (1 g/ml) was added to the culture medium on day 25 of culture, brown pigments appeared precociously, i.e. within the first two days, in the cells. The brown pigments were identified as melanins by histochemical and electron-microscopic methods. Induction of melanogenesis required continuous treatment with amphotericin B. With the precocious appearance of melanins, tyrosinase activity increased rapidly. This rapid increase in tyrosinase activity was inhibited by the addition of phenylthiourea or diethyl-dithiocarbamate. It was not enhanced by iodoacetamide, but was blocked by a low concentration of cycloheximide or actinomycin D. These findings indicate that amphotericin B induces de novo synthesis of tyrosinase rather than activation of pre-existing tyrosinase.     

Formulation of a lactose-free, low-cost culture medium for the production of β- D-galactosidase by Kluyveromyces marxianus

A lactose-free, low-cost culture medium for the production of -d-galactosidase by Kluyveromyces marxianus was formulated. At high aeration rates (2.2 vvm) and concentrations of 100 g sugar cane molasses l^–1 as carbon source and 100 g corn steep liquor l^–1 as vitamin and nitrogen source an enzyme production of 708 U l^–1 h was achieved. This was 20% higher than using a medium that contained lactose which is considered the primary inductor of -d-galactosidase synthesis.     

MSH binding in bomirski amelanotic hamster melanoma cells is stimulated by L-tyrosine

Bomirski Ab amelanotic melanoma cells have recently been shown to undergo striking phenotypic changes when precursors of the melanogenic pathway, L-tyrosine and L-dopa, are added to the culture medium. The changes include increased tyrosinase activity andde novo synthesis of melanosomes and melanin. L-tyrosine and L-dopa appeared to elicit these responses through separate but overlapping regulatory pathways. Here we show an additional effect of L-tyrosine: stimulation of MSH binding capacity. Cells cultured for 24–48 hours in the presence of 200 M L-tyrosine display a 3–4 fold increase in their ability to bind¹²⁵l--MSH. L-dopa did not stimulate MSH binding under the same conditions. In control experiments neither L-tyrosine nor L-dopa had any effect on insulin binding. The amelanotic cells respond to MSH with increased dendrite formation, increased tyrosinase activity without melanin production, and decreased growth rate.     

Temporal and spatial changes of cellulose synthesis inClosterium acerosum (Schrank) Ehrenberg during cell growth

The amount and distribution of wall microfibril synthesis were investigated in the cell-division cycle ofClosterium acerosum. Electron-microscopic examination and a methylation analysis of alkali-extracted wall fragments showed that alkali-extracted wall was mainly composed of microfibrils and that the microfibrils ofC. acerosum were 4-linked glucans, i.e., cellulose. Cellulose synthesis was measured as incorporation of¹⁴C, fed to cells as NaHCO₃, into extracted wall fragments. Extensive cellulose synthesis was coincident with septum formation, continued for more than 6 h and then ceased. It was found by microautoradiography that cellulose synthesis after cell division was essentially restricted to the expanding new semicells. Such a restricted distribution of cellulose synthesis was maintained for more than 6 h after septum formation, i.e., for more than 2 h after the cessation of expansion; afterwards, cellulose synthesis in some, but not all, cells became extended to the old semicells, and then ceased. Considerable cellulose synthesis also took place in the band-like expanding part of non-divided cells, indicating that cell division was not necessarily required for the induction of cellulose synthesis and the latter was coupled with cell expansion. Extension of cellulose synthesis to old semicells was brought about in divided cells by treatment with 3 mM colchicine, 28 M vinblastine, 50 M isopropyl-N-phenylcarbamate or 1 M isopropyl-N(3-chlorophenyl)carbamate, indicating that microtubules are involved in the limitation of cellulose synthesis to the new semicells.Abbreviations CIPC isopropyl-N(3-chlorophenyl)carbamate - DPO 2,5-diphenyloxazole - IPC isopropyl-N-phenylcarbamate     

Role of melanin in fungal biosorption of tributyltin chloride

Summary Intact biomass of an albino and a melanic strain of Aureobacidium pullulans, as well as purified melanin from the latter strain, was capable of tributyltin chloride (TBTC) removal from solution. Melanized biomass had a greater biosorptive capacity than albino biomass, this difference being attributable to the presence of melanin. Purified melanin had a large capacity for TBTC biosorption, the calculated maximum uptake capacity, q _e, being approximately 35 mmol (g dry wt)^–1. TBTC biosorption by intact biomass and melanin obeyed the Langmuir adsorption isotherm over the concentration range used, and was relatively unaffected by external pH between pH 3.5 and 6.5: an approximate 20% decrease in TBTC biosorption resulted at external pH 2.5. A TBTC concentration of 0.3 M in growth medium resulted in a lag period which was longer with the albino strain (approximately 50 h) than with the pigmented strain (approximately 25 h). The addition of melanin to TBTC-containing growth media resulted in a reduction in toxicity and attainment of higher cell yields. The applied and environmental significance of these interactions are discussed. Offprint requests to: G. M. Gadd     

Strategies for the selection and characterization of aluminum-resistant variants from cell cultures of Nicotiana plumbaginifolia

The development of strategies for selecting and characterizing aluminum-resistant variants from Nicotiana plumbaginifolia Viv. cell cultures is described. Plated cells, smeared callus, in-vitro-grown shoots, and seedlings of wild-type N. plumbaginifolia all showed similar responses to Al, with total growth inhibition at or above 600 M Al. The strict control of both cell density and aggregate size is important in selection experiments for total inhibition of the growth of wild-type cells. Two approaches for the selection of Al-resistant variants were used. In a direct method, cells were plated onto medium containing 600 M Al which inhibited growth and chlorophyll synthesis in wildtype cells. A double selection strategy based on both cell growth and greening was used to isolate 29 Al-resistant variants. In the other approach, a rescue method, suspensions were cultured for 10 d in medium containing 600 M Al, then plated onto standard medium for recovery of survivors. Using this strategy, 217 Al-resistant variants were selected. After six to twelve weeks of growth in the absence of Al, each variant was cloned and reselected from single cells. Al resistance was retained in 31% and 51% of the variants selected by the direct and rescue strategies, respectively. Seedling segregation data are presented for the progeny (selfed and backcrossed) of plants regenerated from one of the variants and are consistent with those expected for a single dominant mutation.     

The differential effects of TCA-cycle acids on the growth of plant cells cultured in liquid media containing various nitrogen sources

Alfalfa (Medicago sativa L. cv. Canadian No. 1), tobacco (Nicotiana tabacum L. var. humilis) and wheat (triticum monococcum L.) cells were grown in a defined, liquid medium containing either ammonium sulfate, L-glutamine or potassium nitrate as the sole nitrogen source, and the effects of the tricarboxylic-acid (TCA) intermediates, citrate and -ketoglutarate (5, 10, 15 mM), on the growth (dry-weight increase) of these cells was observed. The three cell suspension cultures exhibited a different growth response to the TCA-cycle intermediate supplied, depending upon the concentration of the additive and the nitrogen source. Citrate (5 mM) greatly enhanced growth of alfalfa and wheat cells in an ammonium-based medium but was less effective at higher concentrations, and in the case of alfalfa cells markedly inhibited growth. Tobacco cell growth was inhibited by all citrate concentrations tested. In contrast, all concentrations of -ketoglutarate used stimulated the growth of all three cell cultures in an ammonium-based medium. Alfalfa and wheat cells grown in an L-glutamine-based medium were influenced by citrate in a manner similar to that in ammonium-based medium. The growth of tobacco cells was slightly enhanced by 5 mM citrate but inhibited by higher concentrations. -Ketoglutarate, at all concentrations tested, was stimulatory to the growth of the cells of all three species in a glutamine-based medium, except for alfalfa cells which were inhibited at 15 mM. Both TCA-cycle acids inhibited the growth of alfalfa and tobacco cells grown on a nitrate-based medium whereas the growth of wheat cells was almost unaffected.     

Response to auxin by cells ofRiella helicophylla during reversible arrest in different cell-cycle phases

When cells of the unistratose meristem ofRiella helicophylla (Bory et Mont.) Mont. are reversibly arrested at G1/S transition by treatment with the inhibitor of thymidylate synthase 5-fluorodeoxyuridine, with the inhibitor of DNA polymerase , aphidicolin, or with an inhibitor of late DNA synthesis, 5-aminouracil, they continue to expand. Simultaneous supply of auxin enhances cell expansion, while simultaneous addition of the auxin antagonistp-chlorophenoxyisobutyric acid prevents cell enlargement. When the meristematic cells are reversibly arrested during G1 phase by treatment with chlorsulfuron, an inhibitor of acetolactate synthase, cell size remains unchanged, but it increases when auxin is supplied simultaneously. Simultaneous application of chlorsulfuron during treatment with 5-fluorodeoxyuridine, aphidicolin or 5-aminouracil, prevents cell expansion. After recovery from 5-fluorodeoxyuridine, aphidicolin or 5-aminouracil treatment, the cellular pattern of the meristem is severely disturbed, while in combination withp-chlorophenoxyisobutyric acid or chlorsulfuron, meristem differentiation is almost unaffected. During reactivation of divisional functions in mature cells induced by isolation of tissue fragments, blockage of DNA synthesis by aphidicolin causes an augmentation of rhizoid initials which are characterized by enhanced RNA synthesis. Exogenous supply of auxin is required for outgrowth of these rhizoid initials, while, in untreated fragments, auxin for rhizoid growth is provided probably by the dividing cells. When reactivation of divisional functions in tissue fragments is reversibly inhibited by chlorsulfuron, no changes in the cells are discernible and application of auxin has no effect; after release from blockage the cells regenerate like those in untreated fragments. The results suggest that the phases of the cell cycle differ with regard to auxin synthesis and competence to respond to auxin. Probably, during cycle inhibition at G1/S or S a rising auxin level causes disintegration of cell-cycle events.Abbreviations APH aphidicolin - 5-AU 5-aminouracil - CS chlorsulfuron - 5-FdUrd 5-fluorodeoxyuridine - PCIB p-chloro-phenoxyisobutyric acid Part of doctoral thesis, University of Kassel, GermanyI thank Professor Luise Stange (this Institute) for her suggestions and many stimulating discussions. This research was supported by a scholarship of the Otto-Braun-Fonds and by a grant of the Deutsche Forschungsgemeinschaft to Professor Stange.     

Brevibacterium divaricatum: Influence of nutritional conditions on the wall composition and release of uncross-linked peptidoglycan chains into medium

Purified cell walls, originating from penicillin-treated (3 g/ml, 1 h) and-untreated Brevibacterium divaricatum cells grown on complex (CM) and glucose minimal medium with (MM) or without (Ca-free MM) calcium carbonate, were isolated by two procedures. Electron micrographs and chemical analysis revealed no differences between identically isolated walls with respect to the presence or absence of either penicillin or calcium carbonate in the glucose growth medium. On the contrary, the appearance and peptidoglycan content of the walls was greatly dependent on the procedure used for their isolation and the walls isolated from the cells grown on complex medium contained more materials other than peptidoglycan. It was shown that the presence of calcium carbonate in the glucose minimal medium was essential for accumulation of large amounts of peptidoglycan chains into the medium. Penicillin-induced interruption of cell wall synthesis was prerequisite for manifestation of the calcium carbonate stimulating effect.Abbreviations CM complex medium - MM chemically defined minimal medium based on glucose and containing calcium carbonate - Ca-free MM MM modified only by the omission of calcium carbonate - ET-walls Enzyme treated walls - FPR-walls French press-ruptured walls     

Cellular immunity in an annelid (Nereis diversicolor,Polychaeta): production of melanin by a subpopulation of granulocytes

Summary We attempted to identify the nature and origin of the pigment produced by the marine worm Nereis diversicolor in order to isolate, in inert brown capsules, foreign objects introduced into its body cavity. This brown pigment, characterized by cytochemical techniques, could be a melanin. The activity of the enzyme phenoloxidase responsible for melanin biosynthesis was detected by enzyme cytochemistry techniques in vacuoles and the Golgi apparatus of coelomocytes activated by the presence of foreign bodies. Morphological techniques combined with a monoclonal immunological probe enabled us to establish that the G₂ granulocytes contain both the precursor of the pigment in dense bodies and the capacity for phenoloxidase synthesis when activated to encapsulate foreign bodies. The G₂ granulocyte may therefore be compared to a melanocyte in which melanin is not stored as in mammals, but immediately extruded following synthesis in the form of a thick fluid.Abbreviations ACTH adrenocorticotropic hormone - Dopa L-3,4 dihydroxyphenylalanine - FITC fluorescein isothiocyanate - G ₁, G ₂, G ₃ granulocyte of types 1, 2, 3 - MSH melanocyte-stimulating hormone - proPo prophenoloxidase     

Cell wall-lytic activity in Chlorella fusca

The soluble fraction of homogenates of synchronous Chlorella fusca was tested for carbohydrate-lyzing activities. With isolated cell walls and -1,4-mannan or carboxymethyl cellulose as substrates, a sharp increase in activity occurred shortly before release of the daughter cells followed by a decline during release. The lytic activities were partially purified by ammonium sulphate precipitation and analyzed by gel filtration on a calibrated column. Apparent molecular weights were 27,000 for cell wall autolysin(s) and -1,4-mannanase, 36,000 for carboxymethyl cellulase and 70,000 for another -1,4-mannanase. Incubation of isolated cell walls with an enzyme preparation purified by ammonium sulphate precipitation resulted in release of up to 70% of the cell wall carbohydrate as monosaccharide, predominantly mannose and glucose. The carbohydrate released in vivo into the culture medium shortly before and during liberation of the daughter cells consisted largely of polymeric material with rhamnose, fucose and mannose as main constitutents. Upon poisoning the cells with NaN₃ or carbonyl cyanide p-trifluoromethoxy-phenylhydrazone, however, a monosaccharide fraction consisting of mannose and glucose was predominant in the medium. It is suggested that the major products of cell wall lysis in vivo are monosaccharides which are rapidly taken up and metabolized by the developing daughter cells in an energy-dependent manner.     

Preferential synthesis of plastid DNA and increased replication of plastids in cultured tobacco cells following medium renewal

During the culture of tobacco BY 2 cells derived from Nicotiana tabacum L. cv. Bright Yellow 2, morphological changes of plastid (pt) nucleoids and their replication were examined by fluorescence microscopy after staining with 46-diamidino-2-phenylindole. Upon transfer to fresh medium, the fluorescence intensity originating from pt nucleoids increased markedly. Copy numbers of ptDNA per cell calculated from the quantitative data by super-sensitive microspectroscopy increased 11-fold within 1 d of culture to reach 11 000, then decreased gradually to 1 000 after one week of culture. Autoradiography by labelling with [³H]thymidine showed that DNA synthesis in plastids occurred exclusively during the first day of culture, whereas nuclear DNA synthesis was observed from the first to the sixth day of culture. Replication of plastids was most frequently observed on the second day. Thereafter the formation of starch granules predominated in plastids up to the fifth day of culture, but the starch granules disappeared in the stationary-phase cells. The meaning of such preferential synthesis of ptDNA upon transfer to fresh medium is discussed in relation to the interaction between plastids and nuclei.Abbreviations pt plastid - DAPI 4,6-diamidino-2-phenylindole     

Structural analysis of the cell walls regenerated by carrot protoplasts

A procedure was developed to isolate protoplasts rapidly from carrot (Daucus carota L. cv. Danvers) cells in liquid culture. High purity of cell-wall-degrading enzymes and ease of isolation each contributed to maintenance of viability and initiation of regeneration of the cell wall by a great majority of the protoplasts. We used this system to re-evaluate the chemical structure and physical properties of the incipient cell wall. Contrary to other reports, callose, a (1 3)-d-glucan whose synthesis is associated with wounding, was not a component of the incipient wall of carrot protoplasts. Intentional wounding by rapid shaking or treatment with dimethyl sulfoxide initiated synthesis of callose, detected both by Aniline blue and Cellufluor fluorescence of dying cells and by an increase in (1 3)-linked glucan quantified in methylation analyses. Linkage analyses by gas-liquid chromatography of partially methylated alditol-acetate derivatives of polysaccharides of the incipient wall of protoplasts and various fractions of the cell walls of parent cells showed that protoplasts quickly initiated synthesis of the same pectic and hemicellulosic polymers as normal cells, but acid-resistant cellulose was formed slowly. Complete formation of the wall required 3 d in culture, and at least 5 d were required before the wall could withstand turgor. Pectic substances synthesized by protoplasts were less anionic than those of parent cells, and became more highly charged during wall regeneration. We propose that de-esterification of the carboxyl groups of pectin uronic-acid units permits formation of a gel that envelops the protoplast, and the rigid cellulose-hemicellulose frame-work forms along with this gel matrix.Abbreviations DEAE Diethylaminoethyl - DMSO dimethyl sulfoxide - ECP extracellular polymers - EDTA ethylenediaminetetraacetic acid - HGA nomogalacturonan - RG rhamnogalacturonan - Tes N-tris(hydroxymethyl)methyl-2-amino-ethanesufonic acid - TFA trifluoroacetic acid Journal paper No. 11,776 of the Purdue University Agriculture Experiment Station     

Modification of Drosophila cell surfaces by concanavalin A

Summary The effect of Con A on the surface morphology of cultured cells of Drosophilia melanogaster growing on coverglasses was examined by scanning electron microscopy. With low lectin concentrations (5–10g/ml) surface filaments disappeared and the cells flattened and spread against the glass surface. Cytoplasmic fusion bridges were observed in areas where cells made contact. Concentrations of Con A ranging between 50–500 g/ml caused cell shrinkage and surface distortions without cell flattening and filament loss. These morphologic effects were not apparent if Con A binding sites were blocked by preincubation with -methyl-D-mannopyranoside before application to the cell cultures. However, once the Con A-mediated changes were in effect, the cells failed to show recovery when they were returned to growth medium and a majority of the cells on the coverglasses degenerated. Presumably the cells whose morphology appears unaffected by Con A treatment are the survivors that repopulate cultures returned to growth medium.Supported by Grants CA-12600 and CA 16619 awarded by the National Cancer Institute, DREW and in part by NIH Biomedical Sciences Grant No. RR-07050. CAA's participation in this project was supported by Training Grant No. 5T01-GM-71-17We wish to thank Dr. Imogene Schneider for providing the cell lines     

Effects of growth temperature upshift on cell cycle progression in <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis>

Cell cycle progression of Cryptococcus neoformans was studied for cells grown exponentially at 15°, 24°, and 30°C. Except for speed, cell cycle progression was similar. In particular, budding occurred relatively soon after initiation of DNA synthesis at 15°, 24°, and 30°C. After growth temperature was shifted from 15° to 30°C, cells were transiently arrested before initiation of DNA synthesis. Thus, similar to Saccharomyces erevisiae, Start was the main susceptible cell cycle controlling point in C. neoformans. However, after spontaneous release from arrest as above, cells were further arrested in the unbudded state. Thus, the timing of budding was delayed just before the G₂ phase, or even until after entering the G₂ phase, but it was also transient, and 5h after the shift buds emerged relatively soon after initiation of DNA synthesis. Thus, C. neoformans cells can respond adaptively to mild stress by delaying budding. The existence of the second susceptible cell cycle control point, i.e., budding, appears to endow C. neoformans with a unique characteristic of stronger inhibition of multiplication than growth. A model of the C. neoformans cell cycle is also presented.     

The Effect of Phenylalanine on DOPA Synthesis in PC12 Cells

DOPA synthesis from phenylalanine was studied in PC12 cells incubated with m-hydroxybenzylhydrazine, to inhibit aromatic L-amino acid decarboxylase. DOPA synthesis rose with increasing concentrations of either phenylalanine or tyrosine; maximal rates (~55 pmol/min/mg protein for tyrosine; ~40 pmol/min/mg protein for phenylalanine) occurred at a medium concentration of ~10 M for either amino acid. The K_m for either amino acid was about 1 M (medium concentration). At tyrosine concentrations above 30 M, DOPA synthesis declined; inhibition was observed at higher concentrations for phenylalanine (300 M). These effects were most notable in the presence of 56 mM potassium. Measurements of intracellular phenylalanine and tyrosine suggested the K_m for either amino acid is 20–30 M; maximal synthesis occurred at 120–140 M. In the presence of both phenylalanine and tyrosine, DOPA synthesis was inhibited by phenylalanine only at a high medium concentration (1000 M), regardless of medium tyrosine concentration. The inhibition of DOPA synthesis by high medium tyrosine concentrations was antagonized by high medium phenylalanine concentrations (100, 1000 M). Together, the findings indicate that for PC12 cells, phenylalanine can be a significant substrate for tyrosine hydroxylase, is a relatively weak inhibitor of the enzyme, and at high concentrations can antagonize substrate inhibition by tyrosine.     

Shape and size changes induced by taurine depletion in neonatal cardiomyocytes

Summary Taurine is a very important organic osmolyte in most adult cells. Because of this property it has been proposed that large changes in the intracellular content of taurine can osmotically stress the cell, causing changes in its size and shape. This hypothesis was examined by measuring cell dimensions of taurine deficient cardiomyocytes using confocal microscopy. Incubation of isolated neonatal rat myocytes with medium containing 5mM-alanine led to a 55% decrease in intracellular taurine content. Associated with the loss of taurine was a reduction in cell size. Two factors contributed to the change in cell size. First, there was a shift in cell shape, favoring the smaller of the two cellular configurations commonly found in the myocyte cell culture. Second, the size of the polyhedral configuration was reduced after ßalanine treatment. These same two events also contributed to size reduction in cardiomyocytes incubated with medium containing 30mM mannitol. Nonetheless, some qualitative differences exist between cells osmotically stressed by increasing the osmolality of the incubation medium and decreasing intracellular osmolality. The results support a role for taurine in the regulation of osmotic balance in the neonatal cardiomyocyte.     

Organization of heterogeneous mitochondrial DNA molecules in mitochondrial nuclei of cultured tobacco cells

Summary The molecular size of mitochondrial DNA (mtDNA) molecules and the number of copies of mtDNA per mitochondrion were evaluated from cultured cells of the tobacco BY-2 line derived fromNicotiana tabacum L. cv. Bright Yellow-2. To determine the DNA content per mitochondrion, protoplasts of cultured cells were stained with 4,6-diamidino-2-phenylindole (DAPI), and the intensity of the fluorescence emitted from the mitochondrial nuclei (mt-nuclei) was measured with a video-intensified photon counting microscope system (VIM system). Each mitochondrion except for those undergoing a division contained one mt-nucleus. The most frequently measured size of the DNA in the mitochondria was between 120 and 200 kilobase pairs (kbp) throughout the course of culture of the tobacco cells. Mitochondria containing more than 200 kbp of DNA increased significantly in number 24 h after transfer of the cells into fresh medium but their number fell as the culture continued. Because division of mitochondria began soon after transfer of the cells into fresh medium and continued for 3 days, the change of the DNA content per mitochondrion during the culture must correspond to DNA synthesis of mitochondria in the course of mitochondrial division. By contrast, the analyses of products of digestion by restriction endonucleases indicated that the genome size of the mtDNA was at least 270 kbp. Electron microscopy revealed that mtDNAs were circular molecules and their length ranged from 1 to 35 m, and 60% of them ranged from 7 to 11 rn. These results indicate that the mitochondrial genome in tobacco cells consists of multiple species of mtDNA molecules, and mitochondria do not contain all the mtDNA species. Therefore, mitochondria are heterogeneous in mtDNA composition.Abbreviations DAPI 4, 6-diamidino-2-phenylindole - mtDNA mitochondrial DNA - mt-genome mitochondrial genome - mt-nucleus mitochondrial nucleus - ptDNA proplastid DNA - pt-nucleus proplastid nucleus - VIM system video-intensified photon counting microscope system     

Plant tyrosine decarboxylase can be strongly inhibited by l-α-aminooxy-β-phenylpropionate

The effect of tunicamycin, an inhibitor of N-glycosylation of proteins, on growth and on synthesis of DNA and protein was studied in suspension cultures from Nicotiana tabacum and Catharanthus rosea. In the presence of 0.1–1 g · ml^-1 tunicamycin, cell division and DNA synthesis stopped in cells which had been proliferating logarithmically, but protein formation continued. Cytophotometric determination of the nuclear DNA content in Catharanthus cells showed that a cell-cycle arrest had occurred in G1 phase. Metabolic labelling of cells with the glycoprotein precursors glucosamine or mannose was inhibited, too. The results indicate that one or more glycoproteins are needed for the cell to pass through the G1 phase, as was recently postulated for animal and yeast cells.Abbreviations TCA trichloroacetic acid - TM tunicamycin