Conserved and variable regions in the chromosomal and extrachromosomal DNA of halobacteria

Summary Total DNA from Halobacterium halobium and other halobacteria strains is separated into two fractions, FI and FII, which differ in their G+C content. FI DNA, which represents the major part of the genome is highly conserved in all purple-membrane-forming halobacteria. Fraction II (FII) consists in H. halobium of three DNA specimen: (a) the previously isolated plasmid pHH1, (b) a heterogeneous set of ccc-DNA molecules present in the cell in low copies, termed minor-circular DNA (MCD) and (c) a new type of more A-T rich DNA segments (chromosomal islands) which, as described here and by Pfeifer and Betlach (1985), are integrated in FI. Sequences homologous to pHH1 occur only in Halobacterium species closely related to H. halobium (like H. cutirubrum), whereas MCD sequences are present in all purple-membrane-forming halobacteria. The sequences of the newly identified chromosomal islands are only found like pHH1, in Halobacterium species, closely related to H. halobium. Total DNA from square halobacteria exhibits no extended homologies to FI or FII DNA from H. halobium. The only common DNA sequences found in all halobacteria are certain insertion elements (ISH), such as ISH26. Based on these data, halobacteria can be subdivided in at least three major groups.Dedicated to Prof. Dr. F. Lingens to his 60th anniversary     

Micropropagation of Telopea speciosissima R. Br. (Proteaceae). 2: Rhizogenesis and acclimatisation to ex vitro conditions

Conditions affecting rhizogenesis in vitro and ex vitro and subsequent acclimatisation of Telopea speciosissima (waratah) were investigated. Clonal selections were successfully rooted in vitro in agar, on filter paper bridges or using crushed quartz-sand, the last substrate resulting in superior growth of roots. The in vitro substrates were impregnated with half-strength MS, 7.5 gl^-1 sucrose and various concentrations of IBA. For the quartz-sand, an IBA concentration of 50 M was optimal, 70% of microcuttings were rooted. No plantlets rooted in vitro were acclimatised to ex vitro conditions (using mist, fog or humidity tent regimes). Microcuttings (25–45 mm in length) were rooted ex vitro in a fog humidity regime (droplet size <10 m) using an IBA powder dip (3 g IBA kg^-1). Neither a mist nor a humidity-tent regime was suitable for rooting of waratah microshoots ex vitro. A peat and perlite mixture was superior to crushed quartz-sand or potting mix for the rooting of microshoots; this appeared to be related to the air-filled porosity (>20%) of the mixture, measured after the medium was saturated and then drained for 24h. Plantlets must be left under the high humidity regime until shoot growth resumes (four to eight weeks) otherwise plant mortality increase significantly. In vitro-produced leaves abscised between eight and 12 weeks after transfer to ex vitro conditions, indicating that these structures did not acclimatise ex vitro.Abbreviations BA benzyladenine - GA₃ gibberellic acid - IBA indole-3-butyric acid - LSD least significant difference - MS Murashige and Skoog medium     

Visualization of the NMDA recognition site in rat and mouse spinal cord by [3H]CGS 19755in vitro autoradiography

Summary The possibility to visualize the NMDA recognition site with [³H]CGS 19755in vitro autoradiography was evaluated in rat brain and spinal cord sections and thereafter used to study the distribution of the NMDA recognition site in rat and mouse spinal cord. The [³H]CGS 19755 binding was concentrated to the dorsal horn, in particular to the substantia gelatinosa. Notable binding was also seen in the intermediate area and ventral horn, while some binding was observed in the funiculi. No major differences were seen in [³H]CGS 19755 binding at various levels of the rat or mouse spinal cord, although a more dense binding was seen in the mouse. A similar distribution of the [³H]CGS 19755 specific binding and the NMDA receptor associated ion-channel site, labeled with [³H]TCP, was found in the mouse spinal cord. Taken together, our data indicate that the NMDA recognition site can be visualized in rat and mouse spinal cord byin vitro [³H]CGS 19755 autoradiography.Abbreviations NMDA N-methyl-D-aspartate - CGS 19755 Cis-4-phosphonomethyl-2-piperidine carboxylic acid - D-AP5 D(—)-2-Amino-5-phosphonopentanoic acid - TCP N-(1-2-thienylcyclohexyl)-3,4-piperidine - MK-801 (±)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate - AMPA -Amino-3-hydroxy-5-methyl-isoxazolepropionic acid - kainate 2-Carboxy-3-carboxymethyl-4-isopropenyl pyrrolidine - CGP 39653 D,L-(E)-2-amino-4-propyl-5-phosphonopentenoic acid     

In vivo andin vitro methylation of lysine residues ofEuglena gracilis histone H1

We have earlier identified and purified two protein-lysine N-methyltransferases (Protein methylase III) fromEuglena gracilis [J. Biol. Chem.,260, 7114 (1985)]. The enzymes were highly specific toward histone H1 (lysine-rich), and the enzymatic products were identified as -N-mono-, di- and trimethyllysines. These earlier studies, however, were carried out with rat liver histone H1 as thein vitro substrate. Presently, histone H1 has been purified fromEuglena gracilis through Bio-Rex 70 and Bio-Gel P-100 column chromatography. TheEuglena histone H1 showed a single band on SDS-polyacrylamide gel electrophoresis and behaved like other histone H1 of higher animals, whereas it had a much higherR _f value than the other histones H1 in acid/urea gel electrophoresis. When theEuglena histone H1 was [methyl-³H]-labeledin vitro by a homologous enzyme (one of the twoEuglena protein methylase III) and analyzed on two-dimensional gel electrophoresis, three distinctive subtypes of histone H1 were shown to be radiolabeled, whereas five subtypes of rat liver histone H1 were found to be labeled. Finally, by the combined use of a strong cation exchange and reversed-phase Resolve C₁₈ columns on HPLC, we demonstrated thatEuglena histone H1 contains approximately 9 mol% of -N-methyllysines (1.40, 1.66, and 5.62 mol% for -N-mono-, di- and trimethyllysines, respectively). This is the first demonstration of the natural occurrence of -N-methyllysines in histone H1.     

Uteroferrin contains complex and high mannose-type oligosaccharides when synthesized in vitro

Mature uteroferrin (Uf; M = 35,500) is a progesterone-induced acid phosphatase secreted by the pig uterus. It contains a single, unphosphorylated, high mannose-type oligosaccharide. Endometrial explants cultured in vitro secrete Uf with a M of 37,000 (37k Uf) having phosphorylated high mannose oligosaccharides. In this report we demonstrate that 37k Uf contains two N-linked oligosaccharides which are a mixture of complex and high mannose-type oligosaccharides. The complex-type glycopeptides are biantennary and a portion may be fucosylated on the GlcNac of the chitobiose core proximal to the peptide. Only a portion of the high mannose-type oligosaccharides are phosphorylated. The remainder appear to be typical Man_6-4GlcNac₂ oligosaccharides found on mature Uf.Abbreviations Uf Uteroferrin - ConA Concanavalin A - WGA Wheat Germ Agglutinin - endoH endo--N-acetylglucosaminidase H - SDS Sodium Dodecyl Sulfate - SDS-PAGE polyacrylamide gel electrophoresis in the presence of SDS     

Effects of 1-aminocyclopropane-1-carboxylic acid and oxygen concentrations on in vivo and in vitro activity of ACC oxidase of sunflower hypocotyl segments

In vivo ethylene production by hypocotyl segments of sunflower seedlings and in vitro activity of 1-aminocyclopropane-1-carboxylic acid oxidase (formerly ethylene-forming enzyme) extacted from the same tissues increase with increasing concentrations of 1-aminocyclopropane-1-carboxylic acid (ACC) and oxygen. ACC oxidase activity follows Michaelis-Menten kinetics. The apparent Km values of the enzyme towards ACC, estimated in vivo and in vitro, are respectively 219 M and 20.6 M. Both Km values towards O₂ are similar, ca 10.6–11.4%. A decrease in concentration in one of the substrates (ACC or O₂) results in an increase in in vivo apparent Km of ACC oxidase for the other substrate. On the contrary, Km values of the enzyme towards ACC or O₂ estimated in vitro are not dependent upon the concentration of the other substrate (ACC or O₂).Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - EFE ethylene-forming enzyme - MACC malonylate 1-aminocyclopropane-1-carboxylic acid - SD standard deviation     

Effect of acute and chronic epinephrine administration on clearance and metabolism of [3H]-epinephrine in trout (Oncorhynchus mykiss)

Several studies have measured the rate of catecholamine clearance, metabolism, and tissue accumulation in fish. However, no information is available on the effect of repeated stress or high circulating catecholamine levels on catecholamine clearance and metabolism. We measured the clearance and metabolism of [³H]-epinephrine (approximately 0.1 g·kg^-1) in SW-acclimated rainbow trout subjected to acute (five injections in 1 day) and chronic (4 days; five injections per day) administration of 4.0 g·kg^-1 epinephrine or saline. In addition, a saturation experiment, where 4.0 g·kg^-1 of unlabelled epinephrine was injected concurrently with [³H]-epinephrine, investigated whether catecholamine clearance and metabolism are affected by high circulatin levels. Neither the rate constants for catecholamine clearance, nor the post-injection proportions of unmetabolised [³H]-epinephrine, deaminated [³H]-epinephrine and O-methylated [³H]-epinephrine were affected by the acute or chronic injection protocols. The concurrent injection of [³H]-epinephrine and 4.0 g·kg^-1 of unlabelled epinephrine resulted in an elevated postinjection ³H:¹⁴C ratio, but increased proportions of O-methylated [³H]-epinephrine and reduced proportions of unmetabolised [³H]-epinephrine. We conclude that in fish (1) catecholamine clearance and metabolism are unlikely to be compromised by repeated exposure to acute stressors; (2) catecholamine extraction and/or metabolism is enhanced when circulating levels are high; and (3) there is a marked capacity to rapidly (minutes) clear and inactivate catecholamines that are released in response to stressful stimuli.Abbreviations CA catecholamines - dpm disintegrations per minute - MAO monoamine oxidase - COMT catecholamine-O-methyltransferase - MOPEG 3-methoxy-4-hydroxyphenylglycol - VMA 3-methoxy-4-hydroxymendelic acid - SW seawater - HPLC high-performance liquid chromatography     

Mast cell growth factor (C-Kit Ligand) in combination with granulocyte-macrophage colony-stimulating factor and interleukin-3:in vivo hemopoietic effects in irradiated mice compared toin vitro effects

In the presence of hemopoietic cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3), mast cell growth factor (MGF; also known as steel factor, stem cell factor, and c-kit ligand) has proven to be a potent hemopoietic regulatorin vitro. In these studies, we examined thein vivo effects of MGF in combination with GM-CSF or GM-CSF plus IL-3. Effects were based on the ability of these cytokines to stimulate recovery from radiation-induced hemopoietic aplasia. Female B6D2F1 mice were exposed to a sublethal 7.75-Gy dose of⁶⁰Co radiation followed by subcutaneous administration of either saline, recombinant murine (rm) MGF (100g/kg/day), rmGM-CSF (100g/kg/day), rmIL-3 (100g/kg/day), or combinations of these cytokines on days 1–17 postirradiation. Recoveries of bone marrow and splenic spleen colony-forming units (CFU-s), granulocyte macrophage colony-forming cells (GM-CFC), and peripheral white blood cells (WBC), red blood cells (RBC) and platelets (PLT) were determined on days 14 and 17 during the postirradiation recovery period. MGF administered in combination with GM-CSF or in combination with GM-CSF plus IL-3 either produced no greater response than GM-CSF alone or down-regulated the GM-CSF-induced recovery. These results sharply contrasted results ofin vitro studies evaluating the effects of these cytokines on induction of GM-CFC colony formation from bone marrow cells obtained from normal or irradiated B6D2F1 mice, in which MGF synergized with GM-CSF or GM-CSF plus IL-3 to increase both GM-CFC colony numbers and colony size. These studies demonstrate a dichotomy between MGF-induced effectsin vivo andin vitro and emphasize that caution should be taken in attempting to predict cytokine interactionsin vivo in hemopoietically injured animals based onin vitro cytokine effects.Abbreviations GM-CSF Granulocyte-Macrophage Colonly-Stimulating Factor - IL-3 Interleukin-3 - MGF Mast Cell Growth Factor - SCF Stem Cell Factor - rm Recombinant Murine - CFU-s Colony Forming Unit-Spleen - GM-CFC Granulocyte Macrophage Colony-Forming Cell - WBC White Blood Cells - RBC Red Blood Cells - PLT Platelets - SLF Steel Factor - G-CSF Granulocyte Colonly-Stimulating Factor - IL-1 Interleukin-1 - IL-6 Interleukin-6 - Epo Erythropoietin - CFC Colony-Forming Cell - Sl Steel - BFU-e Erythroid Burst Forming Units - s.c Subcutaneous - PEG Polyethyleneglycol - PIXY321 GM-CSF/IL-3 Fusion Protein     

Cryopreservation of in vitro-grown shoot tips of apple and pear by vitrification

In vitro-grown shoot tips of apples (Malus domestica Borkh. cv. Fuji) were successfully cryopreserved by vitrification. Three-week-old in vitro apple plantlets were cold-hardened at 5°C for 3 weeks. Excised shoot tips from hardened plantlets were precultured on a solidified Murashige & Skoog agar medium (MS) supplemented with 0.7 M sucrose for 1 day at 5°C. Following preculture shoot tips were transferred to a 2 ml plastic cryotube and a highly concentrated cryoprotective solution (designated PVS2) was then added at 25°C. The PVS2 contains (W/V) 30% glycerol, 15% ethylene glycol and 15% dimethylsulfoxide in medium containing 0.4 M sucrose. After dehydration at 25°C for 80 min, the shoot tips were directly plunged into liquid nitrogen. After rapid warming, the shoot tips were expelled into 2 ml of MS medium containing 1.2 M sucrose and then plated on agar MS medium. Direct shoot elongation was observed in approximately 3 weeks. The average rate of shoot formation was about 80%. This vitrification method was successfully applied to five apple species or cultivars and eight pear cultivars. This method appears to be a promising technique for cryopreserving shoot tips from in vitro-grown plantlets of fruit trees.Abbreviations DMSO dimethylsulfoxide - EG ethylene glycol - PVS2 vitrification solution - LN liquid nitrogen - BA 6-benzylaminopurine - NAA -naphthaleneacetic acid - SE standard error - ABA abscisic acid     

Bacteriophage lambda DNA-dependent synthesis of endolysin in a membranous cell-free system of E. coli

Summary We have previously shown that a membranous cell-free system derived from uninfected penicillin spheroplasts of E. coli transcribes early and late messenger RNA's from DNA.This in vitro system will also transcribe and translate the endolysin gene R of DNA. The enzyme activity that results from in vitro synthesis corresponds to endolysin (a typical late protein) by several criteria.DNA from C_I857 sus R5 ts 9B and C_I857 sus S7 pgal, mutants carrying nonsense mutations in genes involved in the host lysis, are inactive in the synthesis of endolysin with an extract of non permissive cells, although they are fully active with an extract of permissive cells. Furthermore, suppression of these mutations is entirely dependent on addition of supernatant from suppressor strains.The endolysin synthesis from a thermosensitive C_I mutant is observed at 40°C and not at 30°C. This suggests that the product of C_I gene is formed and acts in the in vitro system at 30°C.Enzymatic activity is detected after a 15 min lag period.Membranes and double stranded DNA are absolutely required for the enzyme synthesis. Ribosomes and supernatant highly stimulate the in vitro system.Inhibitors of RNA, DNA and protein synthesis (actinomycine D; cytosine arabinoside; DNA-ase; and chloramphenicol respectively) will prevent endolysin production when added at zero time. If DNA-ase or actinomycine D are added after 20 min of incubation, only partial inhibition of endolysin synthesis occurs. It is therefore concluded, according to our previous observations, that messengers are stable enough to allow enzyme synthesis after delayed addition of the inhibitors in the in vitro system.It appears that there is a complete regulation in the membranous system like in vivo and which starts with the early and late messenger formation and leads to active late protein synthesis.     

Nanoparticle uptake by macrophages in vulnerable plaques for atherosclerosis diagnosis

The composition of atherosclerotic (AS) plaques is crucial concerning rupture, thrombosis and clinical events. Two plaque types are distinguished: stable and vulnerable plaques. Vulnerable plaques are rich in inflammatory cells, mostly only M1 macrophages, and are highly susceptible to rupture. These plaques represent a high risk particularly with the standard invasive diagnosis by coronary angiography. So far there are no non‐invasive low‐risk clinical approaches available to detect and distinguish AS plaque types in vivo. The perspective review introduces a whole work‐flow for a novel approach for non‐invasive detection and classification of AS plaques using the diffusion reflection method with gold nanoparticle loaded macrophages in combination with flow and image cytometric analysis for quality assurance. Classical biophotonic methods for AS diagnosis are summarized. Phenotyping of monocytes and macrophages are discussed for specific subset labelling by nanomaterials, as well as existing studies and first experimental proofs of concept for the novel approach are shown.

In vitro and in vivo detection of NP loaded macrophages (MΦ). Different ways of MΦ labelling include (1) in vitro labelling in suspension (whole blood or buffy coat) or (2) labelling of short‐term MΦ cultures with re‐injection of MΦ‐NP into the animal to detect migration of the cells in the plaques and (3) in vivo injection of NP into the organism.     

Rapid propagation of tuna (Opuntia ficus-indica) and plant establishment in soil

Explants from young joints of mature plants of tuna (Opuntia ficus-indica Mill.) were cultured on Murashige and Skoog (MS) medium containing 8.8 M benzyladenine (BA) and 0.5 M naphthaleneacetic acid (NAA). Shoots produced were utilized as secondary explants. Each shoot was cut longitudinally from apex to base into two explants, and some of these explants were cut transversely into proximal and distal explants. The size and number of shoots produced was affected by size and position of the explant within its source. The shoots were rooted in vitro or ex vitro and plants were successfully established in soil from both rooting methods.Abbreviations AC activated charcoal - BA benzyladenine - IBA indolebutyric acid - MS Murashige & Skoog (1962) medium - NAA naphthaleneacetic acid     

Aminoglycoside-derived cationic lipids as efficient vectors for gene transfection in vitro and in vivo

Background

Cationic lipids are at present very actively investigated for gene transfer studies and gene therapy applications. Basically, they rely on the formation of DNA/lipid aggregates via electrostatic interactions between their cationic headgroup and the negatively charged DNA. Although their structure/activity relationships are not well understood, it is generally agreed that the nature of the positive headgroup impacts on their transfection activity. Thus, we have directed our efforts toward the development of cationic lipids with novel cationic moieties. In the present work, we have explored the transfection potential of the lipophilic derivatives of the aminoglycoside kanamycin A. Indeed, aminoglycosides, which are natural polyamines known to bind to nucleic acids, provide a favorable scaffold for the synthesis of a variety of cationic lipids because of their structural features and multifunctional nature.

Methods and results

We report here the synthesis of a cationic cholesterol derivative characterized by a kanamycin A headgroup and of its polyguanidinylated derivative. The amino‐sugar‐based cationic lipid is highly efficient for gene transfection into a variety of mammalian cell lines when used either alone or as a liposomal formulation with the neutral phospholipid dioleoylphosphatidylethanolamine (DOPE). Its polyguanidinylated derivative was also found to mediate in vitro gene transfection. In addition, colloidally stable kanamycin‐cholesterol/DOPE lipoplexes were found to be efficient for gene transfection into the mouse airways in vivo.

Conclusions

These results reveal the usefulness of cationic lipids characterized by headgroups composed of an aminoglycoside or its guanidinylated derivative for gene transfection in vitro and in vivo. Copyright © 2002 John Wiley & Sons, Ltd.

     

Conformation and folding in histones H1 and H5

Denatured histones H1 and H5 can be readily refolded on salt addition. Their digestion by trypsin leads to limit peptides of about 80 residues having the same nmr and CD spectra as those of the intact parent histones. Scanning microcalorimetry shows that (1) the folded structures of H1 and H5 are located entirely in their limit peptides; (2) both have values of the specific denaturation enthalpy typical for small globular proteins; and that (3) both exhibit a classic “2-state” transition (ΔH = ΔH). The heat-denaturation profiles of H5 measured using intrinsic and extrinsic Cotton effect and side-chain nmr peaks do not coincide at all. Only the intrinsic Cotton effects give a T_m and ΔH close to that from microcalorimetry. We conclude that these proteins exhibit large-scale side-chain motions that precede the macroscopic cooperative transition.     

In vitro propagation of Amaryllis belladonna

Amaryllis belladonna L. plants were multiplied successfully by means of tissue culture techniques. Different plant parts were tested as explant material, but plantlets could only be generated from the twin-scales and immature scapes. These in vitro-formed plantlets were divided into four parts and used for further multiplication. The twin-scale explants had the highest multiplication rate when a medium with 22.2 M benzyladenine and 0.54 M naphthaleneacetic acid was used. The sucrose concentration played an important role in the initiation of new plantlets, and the best results were obtained when a sucrose concentration of 2–3% was used. Anatomical observations were made during the initiation of the new plantlets.Abbreviations BA benzyladenine - NAA naphthaleneacetic acid - Benomyl (methyl [1-[(butylamino) carbonyl]-1H-benzimidazol-2-yl] carbamate) - Folpet (2-[(trichloromethyl)thio]-H-isoindole-1,3(2H)-dione phthalimide(I))     

Kinetic and functional characterization of a membrane-bound NAD(P)H dehydrogenase located in the chloroplasts of Pleurochloris meiringensis (Xanthophyceae)

Using isolated chloroplasts or purified thylakoids from photoautotrophically grown cells of the chromophytic alga Pleurochloris meiringensis (Xanthophyceae) we were able to demonstrate a membrane bound NAD(P)H dehydrogenase activity. NAD(P)H oxidation was detectable with menadione, coenzyme Q₀, decylplastoquinone and decylubiquinone as acceptors in an in vitro assay. K _m-values for both pyridine nucleotides were in the molar range (K _m[NADH]=9.8 M, K _m[NADPH]=3.2 M calculated according to Lineweaver-Burk). NADH oxidation was optimal at pH 9 while pH dependence of NADPH oxidation showed a main peak at 9.8 and a smaller optimum at pH 7.5–8. NADH oxidation could be completely inhibited with rotenone, an inhibitor of mitochondrial complex I dehydrogenase, while NADPH oxidation revealed the typical inhibition pattern upon addition of oxidized pyridine nucleotides reported for ferredoxin: NADP⁺ reductase. Partly-denaturing gel electrophoresis followed by NAD(P)H dehydrogenase activity staining showed that NADPH and NADH oxidizing proteins had different electrophoretic mobilities. As revealed by denaturing electrophoresis, the NADH oxidizing enzyme had one main subunit of 22 kDa and two further polypeptides of 29 and 44 kDa, whereas separation of the NADPH depending protein yielded five bands of different molecular weight. Measurement of oxygen consumption due to PS I mediated methylviologen reduction upon complete inhibition of PS II showed that the NAD(P)H dehydrogenase is able to catalyze an input of electrons from NADH to the photosynthetic electron transport chain in case of an oxidized plastoquinone-pool. We suggest ferredoxin: NADP⁺ reductase to be the main NADPH oxidizing activity while a thylakoidal NAD(P)H: plastoquinone oxidoreductase involved in the chlororespiratory pathway in the dark acts mainly as an NADH oxidizing enzyme.Abbreviations Coenzyme Q₀-2,3-dimethoxy-5-methyl-1,4-benzoquinone - FNR ferredoxin: NADP⁺ reductase - MD menadione - MV methylviologen - NDH NAD(P)H dehydrogenase - PQ plastoquinone - PQ₁₀ decylplastoquinone - SDH succinate dehydrogenase - UQ₁₀ decylubiquinone (2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone)     

Combined effects of photodynamic therapy and irrigants in disinfection of root canals

In this study, the combined effects of photodynamic therapy and irrigants in eradicating common endodontic pathogens are evaluated. Roots of 80 extracted single rooted teeth are divided into 2 groups (1) mechanical flushing; (2) antibacterial irrigation. After cleaning and shaping, they are inoculated with either (A) Streptococcus mutans or (B) Enterococcus faecalis and incubated. They are again subdivided and either only irrigated or irrigated and lased. Dentin shavings are taken from root canal walls and cultured. Statistical analysis using One‐Way ANOVA and Post‐hoc tests are done. The combination eradicated both bacteria. Antibacterial irrigants controlled S. mutans better than PDT (p = 0.041). The combination of PDT and antibacterial irrigation proposed in this study can be used in all primary cases for thorough and reliable disinfection of root canals but may be highly effective in resistant cases like endodontic failures, as E. faecalis is prevalent in such cases.

     

Callus production,suspension culture and in vitro alkaloid yields of Ephedra

The effects of a range of plant growth regulators on callus production in various Ephedra species were examined. Species examined were E. andina, E. distachya, E. equisitina, E. fragilis var, camplyopoda, E. gerardiana, E. intermedia, E. major ssp procera, E. minima and E. saxatilis. All species produced callus on modified MS medium supplemented with 0.25 M kinetin and 5.0 M 2,4-dichlorophenoxyacetic acid or 1-naphthaleneacetic acid. Neither indole-3-acetic acid nor 3-indolebutyric acid induced significant callus formation but the latter maintained growth of established callus cultures in several species. Suspension cultures of several species were established in MS medium supplemented with 0.25 M kinetin and 5.0 M 2,4-dichlorophenoxyacetic acid or 1-naphthaleneacetic acid. Sustained fresh weight doubling times of 70±7h were recorded for cell suspension cultures of E. andina grown in a semi-continous air-lift bubble bioreactor and a minimum doubling time of 56 h was recorded for E. andina in batch culture. It also proved possible to immobilise E. andina batch cultures in sodium alginate beads.Neither parent plants or in vitro cultures of E. distachya, E. fragilis or E. saxatilis produced alkaloids. Trace quantities of 1-ephedrine and trace-0.14% dwt d-pseudoephedrine were produced by in vitro cultures of other species. The ability to produce alkaloid diminished to zero with successive subcultures.Abbreviations Eph 1-ephedrine - Peph d-pseudoephedrine - RGR relative growth rate - KIN kinetin - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IBA 3-in-dolebutyric acid - IAA indole-3-acetic acid