Glycogen in Methanothrix

An intracellular glycogen was purified and characterized from the acetoclastic bacteria Methanothrix str. FE, its average chain length was about 13 glucose residues. Acetyl-CoA was shown to be synthesized by the action of acetate thiokinase; in addition pyruvate synthase, phosphoenolpyruvate synthetase and enzymes of gluconeogenesis were detected in cell extracts. For glycogen synthase activity, both adenosine diphosphate glucose and uridine diphosphate glucose were used as glycosyl donors, apparent K _m were, respectively, 8 M for ADPGlc and 625 M for UDPGLe, at the opposite the V _m were the same for both precursors. This was in accordance with competition experiments and strongly suggested that only one glucosyl transferase was involved and that ADPGlc was the physiological glycosyl donor in Methanothrix str. FE. In addition branching enzyme activity (1-4-glucan-6-glucosyl transferase) was detected in cell extracts.Abbreviations ADPGlc adenosine diphosphate glucose - UDPGlc uridine diphosphate glucose     

Effect of thidiazuron on vegetative tissue-derived somatic embryogenesis and flowering of bamboo Bambusa edulis

Current research on somatic embryogenesis of bamboo uses reproductive tissue as explants. However, it was hard to obtain the explant. Shoots of a local accession (3–4 m high) were used for multiple shoot production. In order to obtain embryogenic callus, nodal and internodal tissues from in vitro plantlets were placed on Murashige and Skoog (MS) medium supplemented with 9.2 M kinetin (KN), 13.6 M 2,4-dichlorophenoxyacetic acid (2,4-D), 0.1% (v/v) coconut milk, and 6% (w/v) sucrose. We studied the effects of sucrose and thidiazuron (TDZ) on callus proliferation. Optimal additives to the MS medium for embryogenic callus proliferation were 0.046 M TDZ, 13.6 M 2,4-D and 3% (w/v) sucrose. TDZ also promoted the germination of bamboo somatic embryos. The germination rate of the somatic embryos exceeded 80% on MS-based medium supplemented with 0.455M TDZ. Naphthaleneacetic acid (NAA) reduced germination. Well-developed plantlets were successfully transferred to soil. There was no albino mutant in subsequent culture. In vitro regenerants and potted plants flowered, but no seeds were produced.     

In vitro culture of Aloe Barbadensis Mill.: Micropropagation from vegetative meristems

A method for rapid and highly effective plant micropropagation from vegetative meristems was established for Aloe barbadensis Mill. Plant micropropagation was achieved culturing apices on medium containing 1.1 M 2,4-dichlorophenoxyacetic acid and 2.3 M kinetin for 15–30 days. High morphogenetic ability was maintained by transferring explants (after 60 days) on media containing 0.11 M 2,4-dichlorophenoxyacetic acid and 2.2 M 6-benzylaminopurine.     

Influence of iron(III) and pyoverdine on extracellular proteinase and lipase production by Pseudomonas fluorescens B52

Factors associated with the production of extracellular lipase and proteinase by Pseudomonas fluorescens B52 during the late-log, early-stationary phase of grown were examined. Active lipase production by resting cell suspensions was observed when cells were harvested during the log phase (A₆₀₀ of 0.3–0.9) Resting suspensions of younger cells (A₆₀₀<0.1) synthesized lipase after a significant lag. Addition of cells of the proteinase-and lipasedeficient mutant P. fluorescens RM14 to B52 cells at low density resulted in stimulation of lipase and proteinase production. Similar results were found using cell-free culture fluid of RM14. Gel filtration on Biogel P2 revealed that the stimulatory factor co-chromatographed with the iron(III) siderophore, pyoverdine. Partially purified pyoverdine stimulated enzyme synthesis at a concentration of 6 M while having no effect on activity of preformed enzyme. Production of pyoverdine and extracellular enzymes was also stimulated by transferrin, a strong iron(III) binding protein. Growth of B52 in deferrated media was limited to 27% of that found with untreated media. Maximum pyoverdine, proteinase and lipase synthesis was obtained at a final iron(III) concentration of 5.75 M. Growth was maximal in 8.75 M iron(III) while synthesis of pyoverdine, proteinase and lipase was reduced to 3.6, 6.6 and 30% respectively in 23.75 M iron(III). Lipase activity in cell-free culture fluid was slightly inhibited by the addition of up to 400 M iron(III) while proteinase activity was unaffected. In dilute cell suspensions, lipase synthesis was more sensitive to iron(III) than was proteinase (50% inhibition at 1.6 M and a maximum of 40% inhibition at 5.0 M, respectively). In the case of lipase, added pyoverdine was able to partially protect enzyme production from the effects of iron(III). The results are consistent with a role for iron(III) in the regulation of extracellular lipase and proteinase synthesis by P. fluorescens.Contribution No. 677 from the Food Research Centre     

Zinc protects against arsenic-induced apoptosis in a neuronal cell line,measured by DEVD-caspase activity

Acute and chronic arsenic exposure results in toxicity in humans and causes many neurological and other manifestations. For the first time the present study reports that zinc decreases arsenic-induced apoptosis and also confirms a single report of apoptosis induced by arsenic in a neuronal cell line. Apoptosis measured by DEVD-caspase activity peaked between 10 M and 20 M of arsenic trioxide. Higher concentrations of arsenic up to 40 M caused increasing cell death with diminishing DEVD-caspase activity. The beneficial effect of zinc was proportional to its concentration with a significant decrease in arsenic-induced DEVD-caspase activity at 50 M and 75 M zinc (P<0.05). This finding may be of therapeutic benefit in people suffering from chronic exposure to arsenic from natural sources, a global problem especially relevant to millions of people on the Indian subcontinent.     

Sisal plant regeneration via organogenesis

Callus was initiated from in vitro grown immature leaf and ex vitro grown mature leaf and rhizome explants of Agave sisalana Perr. ex. Engelm, on MS medium containing 2,4-D (9.05 M) and kinetin (4.6 M) or 2,4-D (9.05 M), kinetin (4.6 M) and CH (1000 mg l^–1) or mod. MS (NH₄NO₃, 1500 mg l^–1) containing 2,4-D (9.05 M) and kinetin (4.6 M). Light was essential for callus formation which, however, was different in three types of explants on three different media compositions. Increasing NH₄ ⁺had a negative impact while addition of CH had a positive impact on callus formation. Shoot regeneration from callus from CH-supplemented medium only was achieved for rhizome and immature leaf tissues. The highest rate of regeneration was obtained with BA (26.6 M) as the sole hormone. Shoot buds g^–1 callus varied according to BA concentrations. Shoot proliferation rate increased on half-strength MS medium containing BA (8.9 M). Microshoots developed on MS medium containing BA (2.22 M) and GA₃ (1.44 M) and finally rooted on MS medium containing IAA (11.42 M). Acclimatized rooted plantlets are growing satisfactorily in ex vitro. This is the first report on plant regeneration via organogenesis of A. sisalana.     

Tissue culture response of Beta germplasm: callus induction and plant regeneration

Callus cultures of 18 sugarbeet (Beta vulgaris) lines, two accessions of B. maritima and a B. macrocarpa accession were initiated from aseptically germinated seeds. Plant regeneration through organogenesis was obtained either on MS or B5 medium containing various concentrations and combinations of naphthaleneacetic acid (NAA), 6-benzylaminopurine (BAP), 2,3,5-triiodobenzoic acid (TIBA) and abscisic acid (ABA). Genotypes differed in their abilities of callus formation and regeneration: seven out of 18 sugarbeet lines, and an accession of B. maritima were capable of regenerating plantlets. Our data also indicated that 2 M TIBA promoted morphogenesis from callus culture in the presence of 5 M BAP.     

Different effects of inorganic and triethyl lead on growth and ultrastructure of lily pollen tubes

Summary Pollen tubes ofLilium longiflorum growingin vitro were treated for 1 h with inorganic lead (Pb) and with triethyl lead (TriEL) and studied by light and electron microscopy. Pb was considerably more toxic in relation to inhibition of pollen tube growth (EC₅₀=6 M Pb) than was TriEL (EC₅₀=60 M TriEL). On the other hand, at almost the entire concentration range tested (25-500 M) TriEL caused aberrant tubes and tube swellings. Pb did not cause tube swellings, even at highly growth-impairing concentrations. Pb (60 M) predominantly affected the ultrastructure of the growing cell walls without impairing the distribution of the cell organelles in the tube tips. In contrast, 50 and 100 M TriEL did not visibly influence cell wall ultrastructure but it severely damaged dictyosomes; 100 M TriEL also disturbed the original order of cell organelles in the tube tips. Cortical microtubules were selectively and completely destructed by TriEL at concentrations (50 M) where no effect on polar organization of the tube tips occurred but they remained unimpaired by 60 M Pb, indicating selective and effective interaction of TriEL with these cell organelles.Abbreviations EC⁵⁰ effective lead concentration causing 50% inhibition of pollen tube growth - MTs microtubules - Pb inorganic lead - TriAL trialkyl lead - TriEL triethyl lead     

Inversion of gravitropism by symmetric blue light on the clinostat

Gravitropic stimulation of maize (Zea mays L.) seedlings resulted in a continuous curvature of the coleoptiles in a direction opposing the vector of gravity when the seedlings were rotated on a horizontal clinostat. The orientation of this response, however, was reversed when the gravitropic stimulation was preceeded by symmetric preirradiation with blue light (12.7 mol photons·m^–2). The fluence-response curve of this blue light exhibited a lower threshold at 0.5 mol·m^–2, and could be separated into two parts: fluences exceeding 5 mol·m^–2 reversed the direction of the gravitropic response, whereas for a range between the threshold and 4 mol·m^–2 a split population was obtained. In all cases a very strong curvature resulted either in the direction of gravity or in the opposite orientation. A minor fraction of seedlings, however, curved towards the caryopsis. Furthermore, the capacity of blue light to reverse the direction of the gravitropic response disappeared with the duration of gravitropic stimulation and it depended on the delay time between both stimulations. Thistonic blue-light influence appears to be transient, which is in contrast to the stability observed fortropistic blue-light effects.This work was supported by the Deutsche Forschungsgemeinschaft.     

The spermatozoon of Oikopleura dioica Fol (Larvacea,Tunicata)

Summary The spermatozoon of Oikopleura dioica is about 30 m long, with a spherical head, about 1 m wide, a 3 m long and 1 m wide midpiece, and a 25 m long tail with a tapered end piece. The head contains a nucleus with the chromatin volume limited to about 0.1 m³. A small acrosome is found in an anterior inpocketing, and a flagellar basal body in a posterior inpocketing of the nucleus. The midpiece contains a single mitochondrion with the flagellar axoneme embedded in a groove along its medial surface. The flagellar axoneme has the typical 9 + 2 substructure, and the basal body the typical 9+0 substructure. A second centriole and special anchoring fibres are absent.     

Resistance to Spodoptera exigua in Apium prostratum

Two Apium accessions were compared with the commercial cultivar Tall Utah 52–70R (A. graveolens [L.]) for resistance to Spodoptera exigua (Hübner)(Lepidoptera: Noctuidae). Oviposition rate was not significantly different between the three genotypes. In all accessions, eggs were usually placed on the upper half of the plants. Implications of this oviposition pattern on S. exigua management in celery are discussed. The wild species A. prostratum ssp prostratum var filiform (A230) showed a significantly higher resistance to S. exigua than 52–70R. The levels of carcinogenic and mutagenic linear furanocoumarins in the commercial cultivar 52–70R (1.41 g/g in the petioles; 5.85 g/g in the leaves) and in the plant accession A. nodiflorum (5.40 g/g in the petioles; 2.99 g/g in the leaves) were far below the concentration reported to produce acute contact dermatitis (18.0 g/g). The levels of furanocoumarins in A. prostratum petioles (186.14 g/g) and leaves (326.45 g/g) were 10 and 18 times higher, respectively, than the concentration known to cause contact dermatitis. However, resistance in A. prostratum was primarily due to non-preference and the linear furanocoumarins did not induce non-preference. Therefore, the resistance shown by this plant accession does not appear to be furanocoumarin-based and may be suitable for transfer to commercial celery for use in S. exigua management.     

Canavanine synthesis in thein vitro propagated tissues ofCanavalia lineata

Maximum shoot induction from stem explants ofCanavalia lineata was obtained with an agar-solidified PC medium containing 10 M benzylaminopurine and 1 M naphthaleneacetic acid. Rooting of thesein vitro produced shoots was achieved with hormone-free PC medium. Canavanine was produced almost exclusively in the leaves and was not detected in the roots ofin vitro propagatedC. lineata. To exclude the possibility of imminent translocation of canavanine from the root to leaf, adventitious roots were induced from leaf explants in PC medium supplemented with 1 M kinetin and 20 M indole-3-acetic acid and subcultured in medium lacking growth regulators, and the roots excised from germinated seedlings were cultured in hormone-free PC medium. All the roots were incapable of accumulation of canavanine. These results suggest that leaves ofC. lineata are the possible site of canavanine synthesis.     

The influence of temperature on glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase and the regulation of these enzymes in a mesophilic and a thermophilic cyanobacterium

The activities and kinetics of the enzymes G6PDH (glucose-6-phosphate dehydrogenase) and 6PGDH (6-phosphogluconate dehydrogenase) from the mesophilic cyanobacterium Synechococcus 6307 and the thermophilic cyanobacterium Synechococcus 6716 are studied in relation to temperature. In Synechococcus 6307 the apparent K _m's are for G6PDH: 80M (substrate) and 20M (NADP⁺); for 6PGDH: 90M (substrate) and 25M (NADP⁺). In Synechococcus 6716 the apparent K _m's are for G6PDH: 550M (substrate) and 30M (NADP⁺); for 6PGDH: 40M (substrate) and 10M (NADP⁺). None of the K _m's is influenced by the growth temperature and only the K _m's of G6PDH for G6P are influenced by the assay temperature in both organisms. The idea that, in general, thermophilic enzymes possess a lower affinity for their substrates and co-enzymes than mesophilic enzymes is challenged.Although ATP, ribulose-1,5-bisphosphate, NADPH and pH can all influence the activities of G6PDH and 6PGDH to a certain extent (without any difference between the mesophilic and the thermophilic strain), they cannot be responsible for the total deactivation of the enzyme activities observed in the light, thus blocking the pentose phosphate pathway.Abbreviations G6PDH glucose-6-phosphate, dehydrogenase - 6PGDH 6-phosphogluconate dehydrogenase - G6P glucose-6-phosphate - 6PG 6-phosphogluconate - RUDP ribulose-1,5-bisphosphate - Tricine N-Tris (hydroxymethyl)-methylglycine     

N-Methylation of quinolizidine alkaloids: an S-adenosyl-l-methionine: cytisine N-methyltransferase from Laburnum anagyroides plants and cell cultures of L. alpinum and Cytisus canariensis

An S-adenosyl-l-methionine (SAM): cytisine N-methyltransferase could be demonstrated in crude enzyme preparations from Laburnum anagyroides plants and cell cultures of L. alpinum and Cytisus canariensis. The transferase specifically catalyzes the transfer of a methyl group from SAM to cytisine. The apparent K_m values are 60 mol l^-1 for cytisine and 17 mol l^-1 for SAM. Other quinolizidine alkaloids, e.g. angustifoline and albine, are N-methylated by only 10–15%. The transferase shows a pH optimum at pH 8.5. It is activated by dithioerythritol and inhibited by thiol reagents and Fe²⁺ and Fe³⁺. The reaction product S-adenosylhomocysteine is a powerful inhibitor of the transferase reaction. Cell cultures of L. alpinum which have an active SAM: cytisine N-methyltransferase and which are able to N-methylate exogenous cytisine in vivo, do not accumulate cytisine or N-methylcytisine to a detectable degree.Abbreviations GLC gas-liquid chromatography - SAM S-adenosylmethionine - TLC thin-layer chromatography     

In vitro shoot regeneration from leaf explants of Roman Chamomile

Murashige and Skoog (1962) medium supplemented with 1.0 to 4.5 M of BA and 1.0 M of NAA induced adventitious bud formation and shoot development in leaf explants of Roman Chamomile. A higher number of adventitious buds was observed at the proximal end of the explants. Plantlets were replicated and multiplied on MS medium supplemented with 2.25 M of BA and 0.6 M of IAA. Plantlets were rooted on MS medium supplemented with 0.5 M of IBA and successfully weaned in vivo. The plants grew to maturity with high uniformity and no morphological signs of somaclonal variation.     

Mutation proximal to the tRNA binding region of the Nicotiana plastid 16S rRNA confers resistance to spectinomycin.

Summary Nicotiana tabacum lines carrying maternally inherited resistance to spectinomycin were obtained by selection for green callus in cultures bleached by spectinomycin. Two levels of resistance was found. SPC1 and SPC2 seedlings are resistant to high levels (500 g/ml), SPC23 seedlings are resistant to low levels (50 g/ml) of spectinomycin. Lines SPC2 and SPC23 are derivatives of the SR1 streptomycin-resistant plastome mutant. Spectinomycin resistance is due to mutations in the plastid 16S ribosomal RNA: SPC1, an A to C change at position 1138; SPC2, a C to U change at position 1139; SPC23, a G to A change at position 1333. Mutations similar to those in the SPC1 and SPC2 lines have been previously described, and disrupt a conserved 16S ribosomal RNA stem structure. The mutation in the SPC23 line is the first reported case of a mutation close to the region of the 16S rRNA involved in the formation of the initiation complex. The new mutants provide markers for selecting plastid transformants.     

The Effect of Lowered Concentrations of Carbon,Nitrogen and Phosphorus Sources on the Growth Dynamics of the R,S, and M Dissociants of Pseudomonas aeruginosa

The effect of lowered concentrations of carbon, nitrogen, and phosphorus sources in the medium on the specific growth rate of the R, S, and M dissociants of the hydrocarbon-oxidizing strain Pseudomonas aeruginosaK-2, culture pH, and the population composition was studied. Within the first 16 hours of cultivation in all of the four media tested, the R, S, and M dissociants have virtually identical . The maximal values of were reached by the 20th h of growth in the basal medium (R and S dissociants) and in the carbon-deficient medium containing 0.4% glucose (M dissociant). The R and M dissociants showed the most rapid decrease in in the nitrogen-deficient medium containing 0.55% NaNO₃. By the end of cultivation in the basal medium, the pH of the R, S, and M cultures decreased to 6.3, 5.3, and 3.3, respectively. In the case of the carbon-deficient medium, the drop in the culture pH was lower. After a 2.5-day incubation of the S dissociant in the phosphorus- deficient medium containing 0.028% NaH₂PO₄· 2H₂O and of the M dissociant in the basal medium supplemented with chalk powder, these dissociants were completely displaced from the media.     

Arsenic exposure alters activity behaviour of key nitrogen assimilatory enzymes in growing rice plants

Rice seedlings when grown in sand cultures for 5–20 days under 25 and 50 M As₂O₃ in the medium showed a marked decline in growth when compared to controls. Increased absorption of arsenic from the medium, against the concentration gradient was observed. Greater localization of absorbed arsenic was noted in roots than in shoots. Rice plants grown for 20 days with 50 mol l^–1 arsenic in the medium accumulated upto 370 mol arsenic kg^–1 dry weight in roots. Increasing levels of As₂O₃ in situ caused a marked decline in the activities of the nitrate assimilatory enzymes nitrate reductase (NR), nitrite reductase (NiR) and glutamine synthetase (GS), whereas an increase in the activities of alanine and aspartate aminotransferases was observed. The activities of aminating (NADH-GDH) and deaminating (NAD⁺-GDH) glutamate dehydrogenases increased at moderately toxic level (25 M) of As₂O₃ whereas a higher As level of 50 M was inhibitory to the enzymes. Addition of 1 M proline in the reaction medium caused significant restoration in As-led loss of NR and GS activities. NR and GS extracted from arsenic exposed seedlings showed higher K _m values compared to the enzymes extracted from control-grown seedlings, whereas GDHs extracted from As-stressed seedlings showed a decrease in K _m. Results suggest that inhibition in the activities of N assimilatory enzymes accompanied with decreased affinity of the enzymes towards their substrates would eventually lead to a marked suppression of N assimilation and impaired growth of rice seedlings in As polluted environment.     

Induction of apoptosis by gemcitabine in BG-1 human ovarian cancer cells compared with induction by staurosporine, paclitaxel and cisplatin

A variety of chemotherapeutic agents induce cell death via apoptosis. We had shown previously that gemcitabine (2,2-difluorodeoxycytidine) induced an atypical apoptosis in BG-1 human ovarian cancer cells; therefore, further studies were conducted to characterize more precisely gemcitabine-induced apoptosis in BG-1 cells compared to a general inducer of apoptosis, staurosporine. BG-1 cells exposed to 0.5, 1.0 and 10 M gemcitabine for 8 h, or staurosporine (1.0 M) for 6 h, exhibited high molecular weight DNA fragmentation (50 kbp); however, only staurosporine treatment produced internucleosomal DNA fragments (200 bp) in a laddered pattern on the agarose gel. Staurosporine (1.0 M) rapidly induced phosphatidylserine plasma membrane translocation that increased linearly with time as measured by annexin V-FITC binding, and similar kinetics were observed for caspase activation by staurosporine in BG-1 cells. In contrast, 10 M gemcitabine increased phosphatidylserine expression in a small fraction of cells (5–10%) vs. untreated controls over the course of 48 h and significant caspase activity was detected within 12 h of drug exposure. Time-lapse video microscopy of BG-1 cells exposed to 1.0 M staurosporine or 10 M gemcitabine for up to 72 h showed that the morphologic changes and kinetics of cell death induced by these agents differed significantly. We also evaluated the apoptosis induced by paclitaxel (a mitotic poison) and cisplatin (an agent not dependent on cell cycle functions) in BG-1 cells by these methods because these drugs are used clinically to treat ovarian cancer. Our findings demonstrate that the kinetics of apoptotic cell death induced by gemcitabine and other chemotherapeutic agents should be taken into account when designing treatment strategies for ovarian cancer.     

Direct regeneration of Drosera from leaf explants and shoot tips

An efficient protocol for the micropropagation of Drosera anglica, D. binata and D. cuneifolia is described. Proliferation was obtained from leaf segments and shoot tips, which served as initial explants. The regeneration capacity of explants was influenced by factors such as nutrient media, concentrations of growth regulators and the type of medium (liquid or solid). The highest number of plants regenerating from D. binata explants was obtained on the growth regulator-free Vacin and Went medium. In the case of D. anglica the highest proliferation rate was obtained on the Fast medium supplemented with 0.05 M 6-benzyladenine (BA) and 0.005 M -naphthaleneacetic acid (NAA), whereas for D. cuneifolia the optimal regeneration medium proved to be 1/2 MS with the growth regulator supplementation estimated at 0.2 M BA and 0.2 M NAA. Liquid media significantly increased the regeneration potential of D. anglica and D. binata explants.