The bactericidal effects of Lactobacillus acidophilus,garcinol and Protykin® compared to clarithromycin,on Helicobacter pylori

Chronic infection with Helicobacter pylori causes peptic ulcers, gastric cancer and lymphoma. We evaluated the inhibitory effects of the probiotic Lactobacillus acidophilus DDS-1J, the antibiotic clarithromycin and the natural antioxidants garcinol and Protykin^® (containing 50% trans-resveratrol) on Helicobacter pylori strain ATCC 49503. The findings of this study indicate that Lactobacillus acidophilus DDS-1J exerts a growth inhibitory effect on H. pylori at a ratio of 1:1 or higher in vitro. In the case of clarithromycin, garcinol and resveratrol, the bactericidal effect is time and concentration dependent. Clarithromycin completely inhibited growth at 62.5 g/ml at 6 h and at 31.5 g/ml at 12 h. For garcinol the highest concentration needed for complete inhibition was 31.5 g/ml at 6 h and 3.9 g/ml after 12 h incubation. For resveratrol, significant inhibition was noted at 1000 g/ml at 12 h only. The bactericidal effect of garcinol was reduced by the addition of resveratrol at all concentrations 125 g/ml at 6 and 12 h. We conclude from this study that Lactobacillus acidophilus DDS-1J inhibits H. pylori at 1:1 and higher ratios. Also, between the two antioxidants, garcinol is much more potent than resveratrol as a bactericidal agent against H. pylori, and that resveratrol may antagonize this effect. Finally, our study showed equivalent or better bactericidal activity of garcinol compared to clarithromycin against H. pylori at 6 and 12 h incubation, indicating a potential role for this antioxidant in treatment for H. pylori infection.     

Procedures for the axenic isolation of conchocelis and monospores from the red seaweed Porphyra yezoensis

In order to maintain axenic seedstock cultures axenically of thecommercially important red seaweed, Porphyra yezoensis, aprocedure was developed for axenic isolation and culture of conchocelis andmonospores. For axenic isolation of the conchocelis, contaminated microalgaewere most effectively removed by filtering contaminated samples through a100-m mesh after sonication. Removal of bacteria and otheralgaewas accomplished using a mixture of 5 agents (0.02% chitosan, 100 gml^–1 GeO₂, 10 gml^–1 ampicillin, 40 gml^–1 kanamycin and 200 gml^–1 streptomycin). Axenic single colonies wereisolatedfrom a semi-solid medium prepared from 1% transfer gel. After collectingmonospores from the 40–50% density layer on a percoll-gradient, removalofbacteria and fungi from the monospores was accomplished using a mixture of 5antibiotics (3.5 g ml^–1 nystatin, 2 mgml^–1 ampicillin, 400 gml^–1 kanamycin, 50 gml^–1 neomycin and 800 gml^–1 streptomycin). Axenic single juvenile blades wereisolated from a semi-solid medium prepared from 0.5% transfer gel.     

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.     

Tissue culture ofKarwinskia humboldtiana—a plant producing toxins with antitumoural effects

Isolated embryos ofKarwinskia humboldtiana were cultured in vitro. The growth of embryos and development to plantlets on woody plant medium supplemented with indole-3-acetic acid 6.10^-2 mol l^–1, gibberellic acid (GA₃) 3.10^-2 mol l^–1, and 6-benzylaminopurine (BA) 2 mol l^–1 was obtained. Multiplication of shoots and rooting of excised shoots has been achieved. Callus formation on modified Murashige-Skoog medium supplemented with 1-naphthaleneacetic acid 10 mol l^–1, GA₃ 14 mol l^–1, and kinetin 5 mol l^–1 on hypocotyls, or on root cultures on medium supplemented with 2.4-dichlorophenoxyacetic acid 10 mol l^–1 and BA 10 mol l^–1 was induced.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - TEM transmission electron microscopy     

Transformation of Atriplex gmelini plants from callus lines using Agrobacterium tumefaciens

Atriplex gmelini plants were regenerated via organogensis from hypocotyl explants. Callus lines were induced from the hypocotyl explants on Linsmaier and Skoog (LS) medium supplemented with 1 M benzyladenine and 5 M -naphthaleneacetic acid in the dark. Shoots were regenerated from the callus lines on LS medium supplemented with 20 M thidiazuron and 0.1 M -naphthaleneacetic acid under a high-intensity light condition (450 mol m^–2 s^–1). The regenerated shoots were rooted on LS medium without growth regulators to obtain fully developed plants. We succeeded in transforming Atriplex gmelini from callus lines using Agrobacterium tumefaciens.     

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.     

Micropropagation of the rare species Stylidium coroniforme and other Stylidium species

The number of plants in the gazetted rare species Stylidium coroniforme was increased through micropropagation. A method was first developed using the common species S. brunonianum. It was found that for both species, rapid propagation could be obtained by excising shoots from sterile seedlings and inducing shoot proliferation on Murashige and Skoog medium supplemented with 1 M BAP. Rooting was achieved using 1 M IBA and over 100 plants of each species were successfully established in soil. Leaf pieces could also be used to initiate cultures. In media with 20–25 M BAP and 1–5 M IBA, leaf pieces of S. brunonianum, S. piliferum, S. caricifolium and S. crassifolium produced adventitious buds, thus providing another method of micropropagation.     

Transient expression in leaf mesophyll protoplasts of Arabidopsis thaliana

Conditions for maximising transient expression of GUS in leaf mesophyll protoplasts of Arabidopsis thaliana ecotype C24 were investigated. It was found that the factors most influencing expression levels, with optimum levels in parenthesis, were plasmid DNA quantity (100 g per 5 × 10⁵ protoplasts), inclusion of carrier DNA (50 g), PEG pH and amount (pH above 6, and total PEG concentration at least 9% w/w) and the topological form of the DNA. Linearised plasmid DNA with long flanking sequences 3 and 5 to the marker gene yielded the highest levels of GUS expression.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - GUS -glucuronidase - MU methylumbelliferone - PEG polyethylene glycol - X-gluc 5-bromo-4-chloro-3-indolyl--glucuronic acid     

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     

Cell wall regeneration by protoplasts of Chlamydomonas

Protoplasts from Chlamydomonas smithii prepared by the action of C. reinhardii gamete autolysine have been studied with respect to cell wall regeneration. Natural protoplasts within sporangia were also investigated for purposes of comparison. In both cases a new cell wall is completed within 2–3 h of the onset of regeneration. The first visible stages of wall regeneration are to be seen after 40–60 min as a fine fringe outside of the plasmalemma. The development of the typical central triplet follows within the next 1 h. Cell wall regeneration is reversibly inhibited by cycloheximide (10g ml^-1) and reversibly disturbed by concanavalin A (50 g ml^-1). Actinomycin D at concentration over 100g ml^-1 also inhibit but the inhibition is irreversible and peculiar membrane effects are observed. Chelators (ethylenediamine tetraacetic acid; ethyleneglycol-bis-aminoethyl ether) and 2-deoxyglucose slightly retard or have no effect on cell wall regeneration.Abbreviations EDTA ethylenediamine tetraacetic acid - EGTA ethyleneglycol-bis(aminoethyl ether) - N,N tetraacetic acid     

Purification and properties of recombinant β-glucosidase of the hyperthermophilic bacterium Thermotoga maritima

A -glucosidase of the hyperthermophilic bacterium Thermotoga maritima has been purified from a recombinant Escherichia coli clone expressing the corresponding gene. The enzyme was found to be a dimer with an apparent molecular mass of approximately 95 kDa as determined by size exclusion chromatography. It was composed of two apparently identical subunits of about 47 kDa (determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis). The enzyme had a bbroadsubstrate specificity and attacked -glucoside, -galactoside, -fucoside, and, to a very small extent, also -xyloside substrates. -Glycosidic bonds were not hydrolysed. Kinetic measurement of the hydrolysis of o-nitrophenyl--d-glucopyranoside (oNPGlc) and o-nitrophenyl--d-galactopyranoside (oNPGal) in the concentration ranges 0.05–20 mm and 0.1–10 mm, respectively, at 75°C resulted in non-linear Lineweaver-Burk and Eadie-Hofstee 3lots whereas cellobiose and lactose did not induce this type of effect. Lactose caused substrate inhibition above 350 mm. The enzyme was optimally active at about pH 6.1. The T. maritima -glucosidase represents the most thermostable -glucosidase described to date. In 50 mm sodium phosphate buffer, pH 6.2, at an enzyme concentration of 50 g/ml, the pure enzyme without additives retained more than 60% of its initial activity after a 6-h incubation at 95°C. Correspondence to: W. Liebl     

Coccidia of Brazilian edentates: Eimeria cyclopei n.sp. from the silky anteater,Cyclopes didactylus (Linn.) and Eimeria choloepi n.sp. from the two-toed sloth,Choloepus didactylus (Linn.)

Summary Eimeria cyclopei n.sp. is described from the silky anteater, Cyclopes didactylus, from Pará State, north Brazil. Undifferentiated oocysts, passed in the faeces, complete sporulation in seven days at 26 to 28°C. Oocysts are ellipsoidal to sub-spherical, with a mean size of 28.1 × 23.6 m: the wall is 1.5 to 2.0 m thick, apparently with an outer thin, colourless membrane and two inner, thicker, striated and yellowish layers. There is no micropyle, oocyst residuum or polar body. The mean measurements of sporocysts are 19.0 × 9.0 m, and they are slightly asymmetrical, elongate pear-shape, with a plug-shaped Steida body projecting beyond the end of the sporocyst. Sporozoites are as long as or longer than the sporocysts: The sporocyst residuum is scattered between sporozoites in younger specimens and becomes condensed into rounded mass in older ones. The endogenous stages occur in the epithelial cells of the ileum, on the lumenal side of the host-cell nucleus. Uninucleate meront, microgamont and macrogamont precursors are recognizable morphologically. Mature meronts are 20.0 × 15.7 m some produce 12 to 20 merozoites which are 8.7 × 2.0 m, and others 10 to 26 merozoites which are 11.4 × 2.0 to 15.0 × 3.0 m. Mature microgamonts which are 27.5 × 24.1 m, produce from 150 to 170 microgametes of 7.1 × 1.0 m: microgametes have two flagella of unequal length. Mature macrogamonts are 28.4 × 24.5 m Eimeria choloepi n.sp. is recorded from the two-toed sloth, Choloepus didactylus, from the same area of Brazil. Undifferentiated oocysts, passed in the faeces, complete sporulation in 23 days at 26 to 28°C. Oocysts with a mean size of 23.0 × 20.3 m, have a wall 2.0 to 2.5 m thick which is composed of two thick, yellowish and striated outer layers and a delicate, colourless inner one. There is no micropyle, oocyst residuum or polar granule. Mature sporocysts with a mean size of 11.3 × 7.1 m, are ellipsoidal to egg-shaped and have a poorly developed Steida body. The sporocyst residuum is composed of a small number of large globules: The sporozoites are longer than the sporocyst and strongly recurved. The endogenous stages occur in epithelial cells of the ileum, on the lumenal side of the host-cell nucleus. Dimorphic meronts produce 8 to 18 merozoites which are either 13.0 × 2.0 m or 13.0 × 3.0 m. Microgamonts produce 50 to 80 microgametes of 8.0 × 1.0 m. Mature macrogamonts are 18.3 × 17.9 m. ac]19820212     

Human HE2 (μB) and μA motifs show the same function as whole IgH intronic enhancer in transgenic mice

In the murine IgH gene intronic enhancer (ENH_iH), two major functional domains were reported. One is the E4/octomer region and another includes the A and B motifs. In the human ENH_iH, it was reported that the HE2, which corresponds to the murine B, and E6 motifs play an important role in an enhancer activity and a tissue-specificity at cellular level. Here we examined thein vivo function of the E6, A and HE2 motifs within the human ENH_iH by using the transgenic mice technique. The A and HE2 motifs together revealed almost the same enhancer function as the whole human ENH_iH, but the E6 motif had lesser enhancer acitivty and tissue-specificity.     

Rooting apple cultivars in vitro: Interactions among light,temperature, phloroglucinol and auxin

Delicious apple (Malus domestica Borkh.) and several of its strains, which have been difficult to root in vitro, were successfully propagated with rooting percentages up to 100%. The combination of treatments used to achieve this result included placing the shoots on rooting medium in the dark at 30°C for the first week of the rooting stage, then moving them to a regime of 16 hr light-8 hr dark at 25°C. The rooting medium contained half strength Murashige and Skoog salts plus 1.2 M thiamine HCl, 0.56 mM myo-inositol, 1 mM phloroglucinol (PG), 1.4 M indolebutyric acid (IBA), 1.3 M gibberellic acid (GA₃), 87.6 mM sucrose, and 7 g l^–1 Difco Bacto agar. Dark treatment applied during the proliferation stage (etiolation) was less effective than one applied at the beginning of the rooting stage. The optimum length of dark treatment during rooting was 4 to 7 days. Increasing the temperature from 25°C to 30°C improved rooting of Delicious, Royal Red Delicious, and Vermont Spur Delicious in the absence of PG but generally had less effect in the presence of PG. Further increase in temperature to 35°C stimulated rooting of Royal Red Delicious but reduced rooting of Vermont Spur Delicious. Transfer of the cuttings to auxin-free medium after 1 week had no effect on percentage rooting and increased the number of roots per cutting for only 1 of 4 cultivars tested and then only in the presence of PG. In general PG stimulated rooting of Delicious and its strains, but had no effect on Golden Delicious.     

Effects of lead and cadmium on chemical composition and total water content of the pupal parasitoid, Pimpla turionellae

The parasitoid Pimpla turionellae L. (Hymenoptera, Ichneumonidae) was fed on Cd, Pb and Cd+Pb-contaminated food (33g Cd, 82g Pb and 33g Cd+82g Pb per gram food fresh weight, respectively). Significant decrease in the total lipid and protein content was found along with an increase in the water content particularly in Cd-contaminated parasitoids.     

Regeneration of plants from callus and embryos of ‘Royal’ apricot

Immature embryos of apricot (Prunus armeniaca L.) cv. Royal with a PF index of 25–100 were used to regenerate plants in vitro using two methods. In the first case, callus was initiated on MS medium with 4.5 M 2, 4-D plus 0.44 M BA and regeneration of shoots from the callus occurred on MS medium with 4.4 M BA plus 1.0 M 2, 4-D. In the second case, adventitious buds were directly regenerated from the cotyledons on MS medium with 4.4 M BA plus 1.0 M 2, 4-D.Abbreviations BA 6-benzyladenine - IBA dole-3-butyric acid - NAA -naphthylacetic acid - 2, 4-D 2, 4-dichlorophenoxyacetic acid - PF (embryo length/seed length) x 100     

An abscisic acid analog inhibits abscisic acid-induced freezing tolerance and protein accumulation, but not abscisic acid-induced sucrose uptake in a bromegrass (Bromus inermis Leyss) cell culture

The application of abscisic acid (ABA), either as a racemic mixture or as optically resolved isomers, increases freezing tolerance in a bromegrass (Bromus inermis Leyss) cell culture and induces the accumulation of several heat-stable proteins. Two stereoisomers of an ABA analog, 23 dihydroacetylenic abscisyl alcohol (DHA), were used to study the role of ABA-induced processes in the acquisition of freezing tolerance in these cells. Freezing tolerance was unchanged in the presence of (–) DHA (LT₅₀ -9°C), and no increase in heat-stable protein accumulation was detected; however, the (+) enantiomer increased the freezing tolerance (LT₅₀ -13°C) and induced the accumulation of these polypeptides. All three forms of ABA increased freezing tolerance in the bromegrass cells, although (–) ABA was less effective than either (+) or (±) ABA when added at equal concentrations. Cells pretreated with 20 or 50 M (–) DHA displayed lower levels of freezing tolerance following the addition of 2.5, 7.5 or 25 M (±) ABA. Full freezing tolerance could be restored by increasing the concentration of (±) ABA to > 25 M. Pretreatment of cells with (–) DHA (20 or 50 M) had no effect on freezing tolerance when 25 M (+) ABA was added. The induction of freezing tolerance by 25 M (–) ABA was completely inhibited by the presence of 20 M (–) DHA. The accumulation of ABA-responsive heat-stable proteins was inhibited by pretreatment with 20 M (–) DHA in cells treated with 2.5 or 7.5M (+) ABA, and in cells treated with 25 M (–) ABA. The accumulation of these polypeptides was restored when (±) or (+) ABA was added at a concentration of 25 M. The analysis of proteins which cross-reacted with a dehydrin antibody revealed a similar inhibitory pattern as seen with the other ABA-responsive proteins. The effects of the various isomers of ABA and DHA on cell osmolarity and sucrose uptake was also investigated. In both cases, (±) and (+) ABA had pronounced effects on the parameters measured, whereas (–) ABA treated cells gave substantially different results. In both sucrose uptake and cell osmolarity, DHA had no significant effect on the results obtained following (±) or (+) ABA treatment. Maximum freezing tolerance was only observed in cells when both heat-stable protein accumulation and sucrose uptake were observed.Abbreviations ABA abscisic acid - DHA 2,3 dihydroacetylenicabscisyl alcohols - DMSO dimethyl sulfoxide - LT₅₀ temperature at which 50% of cells are killed The authors would like to acknowledge the technical assistance of Angela Bollman, Bruce Ewan and Angela Shaw. This work was supported by grants from the Natural Science and Engineering Research Council of Canada to L.V.G. and N.H.L., and a grant from the University of Saskatchewan to R.W.W.     

Plant regeneration from embryo-derived callus of oil palm – the effect of exogenous polyamines

Regeneration in oil palm was achieved through somatic embryogenesis/organogenesis from embryo-derived callus. Callus was induced from mature embryos of the cross 281 (D)×18 (P) on modified MS medium supplemented with 2,4-D (113.12 M) and 2-iP (14.76 M). The embryogenic calluses obtained were transferred to Blaydes medium supplemented with 2,4-D (0.045 M) and one of the following growth regulators: TDZ (4.54 M), zeatin riboside (2.85 M), putrescine (1 mM) and spermine (100 M). Secondary somatic embryogenesis was found to occur in media supplemented with polyamines. The efficiency of formation of somatic embryos, secondary somatic embryos and shoot meristemoids were significantly higher in putrescine containing medium. Histological studies were also undertaken.     

Norway spruce somatic embryogenesis: high-frequency initiation from light-cultured mature embryos

Somatic embryos and rooted plantlets have been regenerated from light-initiated embryogenic callus derived from mature embryos of Picea abies. Under a 16 h photoperiod, mature zygotic embryos were cultured on a modified half-strength Murashige & Skoog medium without NH₄NO₃ and supplemented with 5 mM glutamine, 4.5 M N6-benzyladenine and 10.7 M naphthaleneacetic acid or 10 M 2,4-dichlorophenoxyacetic acid. White translucent embryogenic callus, proliferating from the callusing hypocotyl region after 3 weeks incubation, was isolated from the green non-embryogenic tissue and subcultured for over 12 months. Upon transfer of the embryogenic callus through a specific sequence of media, somatic embryos proceeded to mature, elongating and forming rings of cotyledonary leaves similar to those of zygotic embryos. Transferred to medium without growth regulators, the somatic embryos germinated and produced plantlets with green cotyledons, elongated hypocotyls and primary roots.     

Callus proliferation from the protoplasts of embryogenic cells of Quercus serrata

Embryogenic culture was induced from the immature embryos of Quercus serrata using Marashige and Skoog's medium (MS) containing 0.1 M each of 2,4-d and BAP, and subcultured for seven months before isolation of protoplasts by using 1% Cellulase RS in 0.6 M mannitol solution. Efficient colony formation was obtained when protoplasts were cultured in a liquid MS medium containing 0.6 M mannitol, 3% sucrose and combination of 0.1 M or 1 M each of 2,4-d and BAP. Excluding ammonium nitrate from the MS medium resulted in the decrease of the percentage of colony formation. From colonies, both agar culture and liquid culture were sustained in the MS media without mannitol containing no plant growth regulator, or containing 0.1 M of BAP in combination with 0.1 M or 1 M of 2,4-d.Abbreviations BAP 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - MS medium after Murashige & Skoog (1962).