Effect of Sucrose on Starch Accumulation in and Adventitious Bud Formation on Embryos of Picea abies

Adventitious buds were initiated on embryos of Picea abies (L.)Karst. after a pulse treatment with cytokinin. The initial stagesof bud formation could take place on culture medium lackingsucrose, but sucrose was required for further development ofmeristematic centres into bud primordia and buds. Sucrose atone per cent was optimal for adventitious bud formation. Embryoscultured on media containing sucrose started to accumulate starchduring the first day. Starch accumulation occurred especiallyin the cortex cells where starch grains were frequently presentin the chloroplasts. The starch accumulation increased withhigher sucrose concentrations in the culture medium. Embryoscultured on medium lacking sucrose did not accumulate starchbefore the formation of meristematic centres. Starch accumulationwas never observed in meristematic cells from which adventitiousbud primordia developed. Picea abies (L.) Karst., Norway spruce, adventitious bud, starch accumulation, sucrose concentration     

Mutants of Escherichia coli lacking ribosomal protein L11

Three mutants with ribosomes apparently lacking Protein L11, AM68, AM76, and AM77, were investigated using a variety of immunological techniques to determine whether L11 was indeed lacking. Ouchterlony double diffusion, modified immunoelectrophoresis, and dimer formation on sucrose gradients all gave results indicating Protein L11 was missing from the ribosome in these mutants. Electron micrographs of ribosomes of the mutants were indistinguishable from those of wild type. Ribosomes of AM68, AM76, and AM77, did not bind the antibiotic thiostrepton, but binding was recovered upon reconstitution with wild type Protein L11.     

Characterization of translucent segments observed in an smbA mutant of Escherichia coli

Abstract The smbA gene of Escherichia coli is essential for cell proliferation. The smbA2 mutant shows cold-sensitive colony formation at 22°C. A novel morphological phenotype, formation of a translucent segment at midcell or at a cell pole, was observed by phase-contrastt microscopy at a high frequency in the smbA2 mutant cells incubated in L medium lacking NaCl at 22°C, but not observed in L medium containing 1% NaCl or 20% sucrose at the same temperature. No translucent segment was observed in the wild-type cells in any of the media used. Electron microscopic observation revealed that the translucent segments resulted from the enlargement of a periplasmic space by separation of the inner membrane from the peptidoglycan layer and the outer membrane.     

Burkholderia pseudomallei-induced cell fusion in U937 macrophages can be inhibited by monoclonal antibodies against host cell surface molecules

Burkholderia pseudomallei induces the formation of multinucleated giant cells in cell monolayers. After infection of human macrophage-like U937 cells with B. pseudomallei, addition of monoclonal antibodies against integrin-associated protein (CD47), E-selectin (CD62E), a fusion regulatory protein (CD98), and E-cadherin (CD324) suppressed multinucleated giant cells in a concentration-dependent manner while monoclonal antibodies against other surface molecules did not inhibit fusion despite binding to the cell surface. Flow cytometric analysis showed increased expression of CD47 and CD98, but not CD62E and CD324, upon B. pseudomallei infection. Our data suggest the involvement of specific cellular factors in the process of B. pseudomallei-induced fusion.     

Increased ionic strength: effects on DNA fragmentation and poly(ADP-ribose) polymerase activity in HeLa nuclei

Poly(ADP-ribose) polymerase activity was measured in a crude nuclear fraction isolated from HeLa cells. It was found that the addition of ammonium sulfate or other salts to the standard incubation medium inhibited the formation of poly(ADP-ribose). Through the use of alkaline sucrose density gradients it was also noted that this same increase in ionic strength inhibited the in vitro breakdown of the HeLa DNA. Additional experiments with alkaline sucrose density gradients and deoxyribonuclease I showed that the in vitro activity of poly(ADP-ribose) polymerase is largely dependent upon DNA fragmentation but that DNA fragmentation at least in vitro is not dependent upon the formation of poly(ADP-ribose). These observations imply that this nuclear enzyme is not extremely sensitive to changes in the ionic strength of the reaction media but is affected indirectly, supposedly through changes in the endonuclease activity of the HeLa nuclei. If this proves to be true, then the addition of salt to the incubation medium for poly(ADP-ribose) polymerase could prove to be a valuable tool for the study of ADP-ribosylation reactions.     

Role of sugars in regulating transfer cell development in cotyledons of developing Vicia faba seeds

Summary. Transfer cell formation in cotyledons of developing faba bean (Vicia faba L.) seeds coincides with an abrupt change in seed apoplasm composition from one dominated by hexoses to one in which sucrose is the principal sugar. On the basis of these observations, we tested the hypothesis that sugars induce and/or sustain transfer cell development. To avoid confounding effects of in planta developmental programs, we exploited the finding that adaxial epidermal cells of cotyledons, which do not become transfer cells in planta, can be induced to form functional transfer cells when cotyledons are cultured on an agar medium. Growth rates of cotyledons cultured on hexose or sucrose media were used to inform choice of sugar concentrations. The same proportion of adaxial epidermal cells of excised cotyledons were induced to form wall ingrowths independent of sugar species and concentration supplied. In all cases, induction of wall ingrowths coincided with a marked increase in the intracellular sucrose-to-hexose ratio. In contrast, further progression of wall ingrowth deposition was correlated positively with intracellular sucrose concentrations that varied depending upon external sugar species and supply. Sucrose symporter induction and subsequent maintenance behaved identically to wall ingrowth formation in response to an external supply of hexoses or sucrose. However, in contrast to wall ingrowth formation, induction of sucrose symporter activity was delayed. We discuss the possibility of intracellular sugars functioning both as signals and substrates that induce and control subsequent development of transfer cells. Correspondence and reprints: School of Environmental and Life Sciences, Biology Building, University of Newcastle, Callaghan, NSW 2308, Australia.     

Subacute sclerosing panencephalitis (SSPE): isolation of a defective variant of measles virus from brain obtained at autopsy.

A cytopathic agent causing formation of syncytial giant cells was isolated by co-cultivation of human embryonic lung cells with brain cells obtained at autopsy from a patient with subacute sclerosing panencephalitis. Measles specific intracellular immunofluorescence was detected in syncytial giant cells developed by the agent. Paramyxovirus-like nucleocapsids were observed by electron microscopy in nuclei of the syncytial giant cells. Measles specific immunofluorescence was also detected on the surface of unfixed syncytial giant cells. However, the synycytial giant cells did not produce either virions or hemogglutinin, and did not show hemadsorption of African green monkey red cells. Hence, the newly isolated agent seems to be a defective variant of measles virus, and was designated as the SSPE-"BIKEN" strain.     

Radiation induced formation of giant cells(Saccharomyces uvarum)

X-irradiated yeast cells (Saccharomyces uvarum) grown in liquid media stop mitosis and form giant cells. Chitin ring formation, being a prerequisite for cell separation, was studied by fluorescence microscopy using calcofluor white, a chitin specific dye. Experiments with inhibitors of DNA synthesis (hydroxyurea) and chitin synthesis (polyoxin D) demonstrate chitin ring formation to be dependent on DNA synthesis, whereas bud formation is independent of DNA synthesis and chitin ring formation respectively. Basing on these results the formation of X-ray induced giant cells implies one DNA replication which in turn induces the formation of only one chitin ring between mother cell and giant bud. Obviously no septum can be formed. Thus cell separation does not occur, but the bud already formed, produces another bud demonstrating that bud formation itself is independent of DNA synthesis.     

Autolytic Activity and an Autolysis-Deficient Mutant of Clostridium acetobutylicum

The optimum conditions for autolysis and autoplast formation in Clostridium acetobutylicum P262 have been defined. Autolysis was optimal at pH 6.3 in 0.04 M sodium phosphate buffer, and the bacterium produced latent and active forms of an autolytic enzyme. The ability of cells to autolyze decreased sharply when cultures entered the stationary phase. Autoplasts were induced by 0.25 to 0.5 M sucrose and were stable in media containing sucrose, CaCl₂, and MgCl₂. A pleiotropic autolysis-deficient mutant (lyt-1) was isolated. The mutant produced less autolysin than did the parent P262 strain, and it had an altered cell wall which was more resistant to both its own and P262 autolysins. The mutant formed long chains of cells, and lysozyme was required for the production of autoplasts. Growth of the P262 strain or the lyt-1 mutant was inhibited by the same concentrations of penicillin, ampicillin, and vancomycin. The lyt-1 mutant strain treated with the minimum growth-inhibitory concentration of penicillin autolyzed upon the addition of wild-type autolysin to the autolysis buffer at the same rate as did the untreated P262 strain. Chloramphenicol did not protect the penicillin-treated lyt-1 cells against autolysis enhanced by exogenous wild-type autolysin.     

Radiation induced formation of giant cells in Saccharomyces uvarum

Summary Thin sections of budding yeast cells and giant gells grown after X-irradiation have been examined by electron microscopy. The different steps of cross-wall formation during budding were documented with unirradiated cells. With X-ray induced giant cells cytokinesis was shown to be absent. Neither primary nor secondary septae appeared thus cell separation did not occur. Despite this fact both macromolecular synthesis and bud growth continued, giving rise to the formation of giant cells.     

Giant cell formation produced by laser microbeam irradiation of chromatin in Chinese hamster cells

A pulsed laser microbeam of wavelength 532 nm was used to produce visible small lesions in the nucleoplasm or in the cytoplasm of V79 Chinese hamster cells. Transmission electron microscopy (TEM) of microirradiated nuclei showed that the lesions were produced within the nucleus and comprised between 0.2 and 0.5% of the total chromatin. Serial sections above and below the lesion site did not reveal any detectable chromatin damage, indicating that a visible lesion was restricted to the focal point of the beam. Whereas cells microirradiated anywhere in the cytoplasm showed normal clonal growth with few exceptions, the cells containing nuclear lesions did not enter mitosis at the time of unirradiated controls. Instead they formed giant cells in a high percentage of cases ($\text{72}\text{99}$). The DNA content of these cells was considerably increased suggesting polyploidization. In some cases, division of giant cells was observed resulting in non-viable daughter cells containing micronuclei. Further evidence that the induction of giant cell formation depends on chromatin damage was obtained by microirradiation of chromosomes in anaphase. Here, giant cell formation was observed in the daughter cell which received microirradiated chromatin, whereas microirradiation of cytoplasm between the moving sets of chromosomes did not affect subsequent divisions of both daughter cells. Our data point out that loss of reproductive integrity and giant cell formation can be induced by damage at many sites of the chromosome complement.     

The Arabidopsis thaliana Sucrose Transporter Gene AtSUC4 is Expressed in Meloidogyne incognita-induced Root Galls

The plant parasitic nematode Meloidogyne incognita is as an obligate parasite entirely dependent on the plants solute supply. Therefore, the nematodes induce the formation of several giant cells which are embedded into root galls. At present only little information is available about the solute transfer mechanisms of the plants to supply the induced galls and giant cells and consequently the nematodes. In the present work we could show by phloem-loading experiments that giant cells in the roots of Arabidopsis thaliana are not symplasmically connected to the phloem elements, thus differing considerably form the comparable plant–nematode interaction of Arabidopsis and Heterodera schachtii . Consequently the gene expression of the sucrose transporter AtSUC4 ( AtSUT4 ) was studied during nematode development, and its functionality was shown using RNAi gene silencing lines.     

β-Galactosidase activity in cultured cotton cells (Gossypium Hirsutum L.): A comparison between cells growing on sucrose and lactose

Summary Cotton callus and suspension cultures developed from a cotton variety susceptible toXanthomonas malvacearum (E. F. Sm.) Dow, were grown on chemically defined media that contained one of the carbohydrate sources: 3% sucrose, 3% lactose, 3% maltose, 3% fructose, and 3% glucose. All cells were maintained on a medium with sucrose as the carbohydrate and subsequently transferred to media containing the above carbohydrates. Sucrose was the best carbon source for a high growth rate; however, cells growing on glucose, which was almost as good as sucrose, and cells growing on lactose did not turn brown when they reached the stationary phase of growth. A crude extract from callus tissue growing on lactose has a fivefold increase in β-galactosidase [EC 3.21.23] activity as compared with the extract from callus tissue growing on sucrose. When callus tissue growing on lactose was transferred tomedium containing sucrose, β-galactosidase activity decreased to the level as measured in cells maintained on sucrose. Callus cells growing on a lactose medium showed staining when treated with 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside in which very heavy granular stains appeared. Cells growing on sucrose did not show the histochemical staining. These observations suggest that β-galactosidase is induced in cotton callus tissue that has been transferred from a medium containing sucrose to a medium containing lactose. This is journal article J-3704 of the Oklahoma Agricultural Experiment Station. The research was supported in part by a Presidential Challenge Grant from Oklahoma State University and the Oklahoma Agricultural Experiment Station.     

Morphogenesis in hypocotyl cultures of Digitalis obscura: Influence of carbohydrate levels and sources

Hypocotyl explants of Digitalis obscura L. were grown on Murashige and Skoog medium supplemented with 0.57 μM IAA and 4.40 μM BA. The effects of sucrose, maltose, glucose, galactose or mannitol on their growth and bud formation were investigated. None of the carbohydrate sources tested was superior to sucrose, and best results were obtained with 2.0% (w/v) of this disaccharide. Although mannitol did not support morphogenesis, it had a promotive effect on bud formation when added to 1.0 or 1.5% sucrose-supplied media to give the molar sucrose equivalent to 2.0%. The inhibitory effect of high sucrose concentrations could be duplicated by substituting mannitol for sucrose on a molar basis. Our data suggest a dual role of sucrose as osmotic and energy source in D. obscura hypocotyl cultures.     

Hormonal control of cambial activity and vessel differentiation in Quercus robur

Cambial activity and vessel differentiation of the Quercus robur stem were investigated in relation to concentration of growth regulators and sucrose, seasonal changes in the sensitivity of cambial cells, and axial polarity of the stem. Basipetal efflux of natural auxin was measured in the oak stem cambial region. IAA, GA₃, kinetin and sucrose affected cambial activity and/or initiation of vessel differentiation differently, depending upon concentration. Depending upon the season, kinetin increased or reduced the stimulation of cambial activity caused by IAA and GA₃, but it did not affect the differentiation of vessels. Supply of sucrose in higher concentrations reduced the number of differentiated vessels but did not decrease the stimulation of cambial divisions.Unlike stimulation of cambial activity by GA₃, auxin stimulation of cambial divisions and differentiation of vessels were highly dependent upon stem polarity, 2,3,5-triiodobenzoic acid (TIBA) inhibited formation of vessels, but not cambial activity. The oscillations in basipetal efflux of natural auxin from the cambial stem region of successive 6 mm long sections substantiate the hypothesis that the histogenesis of xylem tissue in ring-porous species is under control of the vectoriat field that is associated with oscillatory phenomena in polar auxin transport.     

Development of pig embryos reconstructed by microinjection of cultured fetal fibroblast cells into in vitro matured oocytes.

Nuclear transfer as originally developed for use in amphibians involved microinjecting a nucleus directly into the cytoplasm of the oocyte. A major mammalian modification has been to use cell fusion to introduce the nucleus. Here we report using a microinjection method to introduce small and medium sized fibroblast cells into mature oocytes. Small cells were more likely to result in nuclear formation (30%) than larger cells (15%; P = 0.013). Small, confluent and serum starved cells resulted in nuclear formation more often (P < 0.048) than did cycling cells. The rate of nuclear formation was not dependent upon the media, (NCSU-23 or TL-Hepes without calcium) nor upon the duration of exposure to the media (1 h to 4 h) after microinjection but before activation. While such treatments did not have an effect on nuclear formation, treatment of parthenogenetically activated oocytes with calcium-free TL-Hepes reduced the percentage of blastocysts (P = 0.068. 11.2% vs. 18.3%) and increased the percentage of morula stage embryos (P = 0.007; 27.6% vs. 15.7%) as compared with culture in NCSU. Finally, small confluent cells were used for nuclear transfer and resulted in two presumptive blastocyst stage embryos [2/128 injected or 2/38 (5.3%) successful injections]. These results show that presumptive blastocyst stage embryos can result from microinjection of fibroblast cells to enucleated oocytes and thus may provide a method to create transgenic knockout animals.     

Studies on the mechanism of the osmoresistance of spores of Bacillus subtilis

AIMS: To determine the reason that spores of Bacillus species, in particular Bacillus subtilis, are able to form colonies with high efficiency on media with very high salt concentrations. METHODS AND RESULTS: Spores of various Bacillus species have a significantly higher plating efficiency on media with high salt concentration (termed osmoresistance) than do log or stationary phase cells. This spore osmoresistance is higher on richer media. Bacillus subtilis spores lacking various small, acid-soluble spore proteins (SASP) were generally significantly less osmoresistant than were wild-type spores, as shown previously (Ruzal et al. 1994). Other results included: (a) spore osmoresistance varied significantly between species; (b) the osmoresistance of spores lacking SASP was not restored well by amino acid osmolytes added to plating media, but was completely restored by glucose; (c) the osmoresistance of spores lacking SASP was restored upon brief germination in the absence of salt in a process that did not require protein synthesis; (d) significant amounts of amino acids generated by SASP degradation were retained within spores upon germination in a medium with high but not low salt; (e) slowing but not abolishing SASP degradation by loss of the SASP-specific germination protease (GPR) did not affect spore osmoresistance; (f) sporulation at higher temperatures produced less osmoresistant spores; and (g) spore osmoresistance was not decreased markedly by the absence of the stress sigma factor for RNA polymerase, sigmaB. CONCLUSIONS: Spore osmoresistance appears as a result of three major factors: (1) specific characteristics of spores and cells of individual species; (2) the precise sporulation conditions that produce the spores; and (3) sufficient energy generation by the germinating and outgrowing spore to allow the spore to adapt to conditions of high osmotic strength; the substrates for this energy generation can come from either the endogenous generation of amino acids by SASP degradation or from the spore's environment, in the form of a readily taken up and metabolized energy source such as glucose. SIGNFICANCE AND IMPACT OF STUDY: These results provide information on the mechanisms of spore osmoresistance, a spore property that can be of major applied significance given the use of high osmotic strength with or without high salt as a means of food preservation.     

Galactosidase activity of lactose-positive Neisseria

The chromogenic substrate o-nitrophenyl-beta-d-galactopyranoside (ONPG) was hydrolyzed by lactose-positive Neisseria. Eight strains of pharyngeal origin were examined. In culture reactions, seven strains resembled Neisseria meningitidis with the exception that they produced acid from 1% (w/v) lactose. An eighth strain (V8) differed in that it did not form acid from maltose or from 1% lactose. However, acid formation was observed in 10% lactose cultures of strain V8, suggesting that entry of lactose occurred by passive diffusion, rather than as a result of permease activity. The enzymes which hydrolyzed ONPG were produced constitutively by the cells of all eight strains. Thus, specific activity in these strains was not increased by prior exposure to lactose, or to two other possible inducers, isopropyl-beta-d-thiogalactoside or methyl-beta-d-thiogalactoside. Study of cell-free extracts of one strain showed that the enzyme was heat-labile, having a half-life of 10 min at 45 C. The enzyme was unstable at low protein concentrations, but it was protected completely or partially when albumin or manganous ions were added. The enzyme appeared to be a typical beta-galactosidase: alpha-galactosides (melibiose and p-nitrophenyl-alpha-d-galactopyranoside) were not hydrolyzed, activity against ONPG was not dependent upon inorganic phosphate, and galactose was released by cleavage of ONPG. ONPG hydrolysis provided a simple and rapid method for detecting lactose-positive Neisseria.     

THE INFLUENCE OF GROWTH SUBSTANCES ON THE INDUCTION OF APOGAMY IN PTERIDIUM GAMETOPHYTES

Experiments employing sequences of three media demonstrated the effect of growth substances on the induction of apogamy. The most effective sequence was 4% sucrose 1–14 days, 4% sucrose plus growth substance 15–28 days, and 0.1% sucrose 29–56 days. In this sequence concentrations of NAA, IAA, and GA promoted apogamous shoot formation. A higher NAA concentration than optimal for shoot formation stimulated apogamous root formation in all medium sequences. Kinetin was without effect or inhibitory to apogamy. Combination of kinetin/GA or GA/NAA concentrations did not increase the apogamous response. One combination of the kinetin/NAA concentrations had a synergistic effect on the apogamous shoot formation. Additions of GA to the synergistic kinetin/NAA combination had an antagonistic effect on the apogamous response.