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Genomic DNA methylation of juvenile and mature Acacia mangium micropropagated in vitro with reference to leaf morphology as a phase change marker.

Genomic DNA methylation was analyzed in Acacia mangium Willd. microshoots micropropagated in vitro from juvenile and mature explants, and in relation to leaf morphology of the microshoots, which is considered a phase change indicator. Based on high performance liquid chromatography (HPLC) analyses, we found more DNA methylation in microshoots exhibiting juvenile leaf morphology (22.4%) than in microshoots of the mature phyllode morphological type (20.7%), irrespective of the age of the source material. Overall, the degree of DNA methylation in A. mangium microshoots was consistent with values reported for other angiosperms. Complementary investigations based on methylation sensitive amplification polymorphism (MSAP) techniques established that, of 1204 fragments revealed by the different primer pairs used, 49 (i.e., 4.08%) were derived from C(5m)CGG methylated sites. Three of these C(5m)CGG sites were exclusive to the juvenile plant material, and three sites were exclusive to the mature source. No fragments were associated specifically with leaf morphology, rather than with plant age. Thus, although the two age classes could not be distinguished based on a quantitative HPLC measure of DNA methylation, qualitative differences existed, as demonstrated by the six age-specific markers identified by MSAP. The reliability of the MSAP data was confirmed on a larger sample of juvenile plant material, which suggested that the total of six methylation markers detected is probably an underestimation of the age-related differences in DNA methylation that may exist between juvenile and mature plant materials.

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Isolation and characterization of flower-specific transcripts in Acacia mangium.

Acacia mangium Willd. is a legume tree species native to subtropical and tropical regions of Asia and Australia. Many features of its flower development are common to other legume tree species. To identify genes involved in its floral development, we constructed a subtractive flower cDNA library against vegetative tissues. The 1123 expressed sequence tags (ESTs) represented 576 unique genes. Macroarray analysis further identified 147 of these genes as specific to the early, late or whole flowering process. Eight percent of these flower-specific genes encode MADS-domain-containing transcription factors and MYB proteins. Four percent encode other transcription factors and 10% encode regulatory proteins such as G proteins, kinases and phosphatases. Flower-specific transcripts for gibberellic acid (GA) synthesis and GA-induced proteins, as well as other stress- and pathogenesis-related genes (9%), implicate their involvement in A. mangium flower development. Eighteen percent of the flower-specific genes encode hypothetical proteins and 18% encode proteins of unknown functions. The RNA blot hybridization confirmed and detailed the expression patterns of selected genes. Functions of the A. mangium flower-specific genes are discussed based on comparison with their Arabidopsis homologues, most of which have been implicated in Arabidopsis floral development. Our work suggests general conservation of floral development in A. mangium and Arabidopsis. Further characterization of the conserved and different flower-specific genes will delineate the flowering process of this important legume tree species and facilitate genetic modification of its reproduction.

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Phenotypic and genotypic characterization of bradyrhizobia nodulating the leguminous tree Acacia albida.

Rhizobial isolates that were obtained from both surface and deep soil samples in the Sahelian and Sudano-Guinean areas of Senegal (West Africa) under Acacia albida trees were compared with representative strains of known rhizobial species and genera. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of proteins was used to determine the taxonomic positions of these organisms and the relationships between isolates obtained from the surface and isolates obtained from deep soil. Most of the isolates belonged to eight electrophoretic clusters containing representative strains of Bradyrhizobium japonicum, Bradyrhizobium elkanii, and Bradyrhizobium sp. Isolates were also characterized by the Biolog system, and the results were compared with the results obtained by SDS-PAGE of total proteins; the level of correlation was very low. DNA-rRNA hybridizations with 16S or 23S rRNA from Bradyrhizobium japonicum LMG 6138T (T = type strain) confirmed that most of the protein electrophoretic clusters belong in the Bradyrhizobium-Rhodopseudomonas rRNA complex. Sequencing of 16S rRNA genes showed that some of the A. albida-nodulating isolates belong to a separate lineage together with representatives of other protein electrophoretic clusters. Other isolates that belong to the same electrophoretic cluster as the type strain of Bradyrhizobium japonicum are considered members of the lineage represented by this type strain. The first lineage is as far removed from Bradyrhizobium japonicum as it is from the genus Afipia, Blastobacter denitrificans, and the genus Rhodopseudomonas. The possible relationship among electrophoretic group, geographic origin, and depth of isolation at a particular site is discussed.

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Sphingomonas phyllosphaerae sp. nov., from the phyllosphere of Acacia caven in Argentina.

Two bacterial strains (FA1 and FA2(T)) were isolated from the phyllosphere of a leguminous tree, Acacia caven, in central Argentina. The strains were Gram-negative, strictly aerobic, rod-shaped, motile and formed yellow-pigmented colonies on nutrient agar. The two-primer RAPD patterns of the two strains were identical, suggesting that they belong to the same species. The complete 16S rRNA gene sequences of the two strains were obtained and comparisons demonstrated that they cluster phylogenetically with the species of the genus Sphingomonas sensu stricto. Strain FA2(T) was most closely related (97.6 %) to Sphingomonas adhaesiva. 16S rRNA gene sequence similarities to all other established Sphingomonas species ranged from 94.4 % (to Sphingomonas echinoides) to 97.6 % (to S. adhaesiva). Strains FA1 and FA2(T) were catalase-positive and oxidase-negative. Aesculin was hydrolysed, gelatin and urea were not. beta-Galactosidase was produced. From 51 compounds tested 21 were used as single sources of carbon. The major respiratory lipoquinone was ubiquinone-10. The predominant cellular fatty acids were 16 : 0, 18 : 1omega7c and 16 : 1omega7c (from summed feature 3). Hydroxy fatty acids 14 : 0 2-OH and 15 : 0 iso 2-OH were present as well (from summed feature 4). The polar lipids detected in strain FA2(T) were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, sphingoglycolipid and two unidentified phospholipids. The DNA G+C content of strain FA2(T) was 61 mol%. DNA-DNA hybridization experiments showed 27.6 % relatedness between strain FA2(T) and S. adhaesiva DSM 7418(T). Based upon phenotypic and molecular evidence, a novel species of the genus Sphingomonas is proposed, Sphingomonas phyllosphaerae sp. nov., with strain FA2(T) (=LMG 21958(T)=CECT 5832(T)) as the type strain.

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Polymerase chain reaction denaturing gradient gel electrophoresis analysis of the N2-fixing bacterial diversity in soil under Acacia tortilis ssp. raddiana and Balanites aegyptiaca in the dryland part of Senegal.

Denaturing gradient gel electrophoresis (DGGE) of amplified nifH gene fragments was used to study the diazotrophic community of soil samples under Acacia tortilis ssp. raddiana (legume tree) and Balanites aegyptiaca (non-legume tree), two dominant plant species growing naturally in the dryland part of Senegal. Samples were taken along transects from the stem up to 10 m distance from it, at depths of 0-0.25 m and 0.25-0.50 m. Sampling was done in the dry season (25 June 1999) and in the rainy season (28 August 1999). The community structure and diversity of the bacterial groups from the different samples was analysed further using different techniques, such as statistical analysis and diversity index evaluation of the band patterns. Diazotrophic diversity was lower under B. aegyptiaca than under A. tortilis ssp. raddiana. Multidimensional scaling (MDS) analysis and ANOSIM tests showed a significant effect of the tree on the diazotroph assemblages. SIMPER analysis showed that the major elements responsible for the dissimilarity are a member of the genus Sinorhizobium, which is characteristic of the samples taken under A. tortilis ssp. raddiana and a member of the cluster Bradyrhizobium for the samples taken under B. aegyptiaca. Forty-four major bands were partially sequenced, yielding 33 different nifH sequences, which were used in phylogenetic reconstructions. Most sequences were affiliated with the alpha- beta- and gamma-proteobacteria. Five nifH sequences were identical to those of Pseudomonas stutzeri, and one sequence showed 100% similarity to that of Azotobacter vinelandii. Four bands were affiliated with the Cyanobacteria and a single one with the Firmicutes. For both trees, there were also clear differences between the samples taken in the dry and rainy seasons. Only for the samples taken under A. tortilis ssp. raddiana was a significant difference found between the two sampling depths.

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Characterisation of rhizobia from African acacias and other tropical woody legumes using Biolog and partial 16S rRNA sequencing.

A Biolog (sole carbon source utilisation) user database of tropical and temperature rhizobial strains was created and used in conjunction with the partial 16S rRNA sequencing method to characterise 12 rhizobial isolates from African acacias and other tropical woody legumes. There was close agreement between the two methods but also some significant discrepancies. A high degree of diversity was shown in the relatively small sample of isolates, with 4 out of 5 of the currently proposed rhizobial genera represented. This is the first time Biolog has shown congruence with genotypic fingerprinting using a wide selection of rhizobial reference and test strains.

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Contact allergy to Australian blackwood (Acacia melanoxylon R.Br.): isolation and identification of new hydroxyflavan sensitizers.

Australian blackwood is known to be an important cause of allergic contact dermatitis in Australia. Previous investigations have shown that 2,6-dimethoxy-1,4-benzoquinone and acamelin are 2 of the responsible, but weak, sensitizers. When these 2 quinones are lacking, which is occasionally found, the wood still possesses allergenic properties. The present re-examination led to the isolation and identification of 4 hydroxyflavans, of which 3 proved to be allergens. Melacacidin, known to be the main constituent of these flavan derivatives in the heartwood, was isolated and its sensitizing capacity in guinea pigs determined. It showed a moderate sensitizing power. Melacacidin occurs abundantly in 125 Australian and 3 African Acacia species.

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Characterization of albumin-acacia complex coacervation.

Complex coacervation between oppositely charged albumin and acacia mixtures has been studied, and the applicability of the various theoretical treatments of complex coacervation (the Voorn-Overbeek, Veis-Aranyi, Nakajima-Sato, and Tainaka theories) to this system has been assessed. Under optimum conditions where maximum coacervate yield occurred, the Voorn-Overbeek theory appeared to apply. However, away from the optimum coacervation conditions, coacervate sol formation was observed, which is in accordance with the Veis-Aranyi and Tainaka theories. Microelectrophoretic measurements were used to determine optimum pH and ionic strength conditions for maximum coacervation, based on the method of Burgess & Carless (1984). The effects of pH and ionic strength on coacervate yield are reported. Around the optimum conditions for maximum coacervation a viscous coacervate phase and a relatively clear equilibrium phase are formed.

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Quinonoid constituents as contact sensitisers in Australian blackwood (Acacia melanoxylon RBR).

Australian blackwood (Acacia melanoxylon RBR) is a valuable commercial timber that since 1925 has been incriminated as being injurious to health. In addition to toxic effects numerous cases of allergic contact dermatitis and bronchial asthma have been observed in woodworkers. Several constituents have been identified in recent years, but none of them could be considered as aetiological factors. Sensitizing experiments performed with blackwood heartwood extracts corroborated the described sensitising properties. Chemical studies showed the occurrence of two or possibly three quinones that produced positive skin responses in the sensitised guinea pigs. The main contact allergens were isolated and identified by x-ray analysis. The first, a yellow quinone, was identified as 2,6-dimethoxy-1,4-benzoquinone while the second, a red quinone, has the structure of 6-methoxy-2-methyl-3,5-dihydrobenzofurano-4,7-dion and was named acamelin. Whereas 2, 6-dimethoxy-p-benzoquinone is already known from natural sources, acamelin is new and belongs to the rate group of naturally occurring furanoquinones. Though the obtained sensitising capacity of A melanoxylon RBR in respect of its quinones is not high, it should be considered as a possible source of allergic contact dermatitis, especially as greater amounts of Australian blackwood may enter European countries in the near future.

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Sporocarps of Pisolithus albus as an ecological niche for fluorescent pseudomonads involved in Acacia mangium Wild - Pisolithus albus ectomycorrhizal symbiosis.

Fresh sporocarps and root and soil samples were collected under a monospecific forest plantation of Acacia mangium in Dagana in Northern Senegal and checked for the presence of fluorescent pseudomonads. No bacteria were detected except from sporocarps collected with adhering soil and hyphal strands. Pisolithus sporocarps were dried at 30 degrees C for 2 weeks, ground, passed through a 2-mm sieve and mixed together. This dry sporocarp powder (DSP) was used to inoculate and form mycorrhizas on A. mangium seedlings in a glasshouse experiment. After 3 months culture, plant growth was increased in the DSP treatment but no ectomycorrhizas were present on the A. mangium root systems; however fluorescent pseudomonads were recorded in the cultural soil. The stimulatory effects on the plant growth were maintained for 6 months. However, fluorescent pseudomonads were no longer detected and 35% of the short roots were ectomycorrhizal. Some of the fluorescent pseudomonad isolates detected after 3 months stimulated the radial fungal growth in axenic conditions. These observations suggest that these bacteria are closely associated with the Pisolithus fructifications and could interact with the ectomycorrhizal symbiosis establishment.

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Effect of feeding Acacia abyssinica and of its extracts given by different routes on rats.

The effect of a diet consisting of 2% and 10% of Acacia abyssinica bark on Wistar rats treated for 6 weeks was examined. A 2% A. abyssinica diet was not toxic to rats. Impairment of growth and hepatonephropathy were observed in rats on a 10% A. abyssinica diet. By whatever route it was administered, either intraperitoneally (i.p.) or orally (p.o.), the ethyl acetate extract in daily doses of 500 mg/kg body weight was the most toxic and lethal to rats and caused hepatonephropathy, widespread hemorrhage and congestion and fibrinous peritonitis following i.p. administration. The aqueous and ethanol extracts i n similar doses to ethyl acetate extract were only lethal to rats when given via the i.p. route. Lesions were accompanied by anemia, leukopenia and alterations in serum AST activity and concentrations of urea, total protein and albumin.

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Acacia concinna saponins. I. Structures of prosapogenols, concinnosides A-F, isolated from the alkaline hydrolysate of the highly polar saponin fraction.

A highly polar saponin mixture from pods of Acacia concinna (Leguminosae) was hydrolyzed with alkali to yield five new triterpenoidal prosapogenols named concinnosides A (6), B (3), C (7), D (4), and E (8), together with four known glycosides, acaciaside, (2), julibroside A1 (10) julibroside A3 (9), albiziasaponin C (5), and their aglycone, acacic acid lactone (1). The structures of these new prosapogenols were elucidated based on spectroscopic means. A less polar saponin fraction from the pods gave spinasteryl glucoside and its dihydro derivative.

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Enhancement of membrane damage by saponins isolated from Acacia auriculiformis.

Acaciaside A and B, two acylated triterpenoid bisglycosides isolated from the funicles of Acacia auriculiformis, are known to have antihelmintic activity. Since the saponins contain a conjugated unsaturated system that is highly susceptible to peroxidation, we investigated the interaction of saponins and membrane using rat liver microsomes as our model. Microsomal membranes were incubated with saponins at 30 degrees C for 2 hr. Following incubation, lipid peroxidation was measured in terms of malondialdehyde and conjugated diene. Our results showed that incubation of microsomal membranes with saponins increased both malondialdehyde and conjugated diene. The results suggest that in our model, saponins enhance the membrane lipid peroxidation.

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Effect of aqueous extract of Acacia nilotica ssp adansonii on milk production and prolactin release in the rat.

In view of the traditional belief that Acacia nilotica ssp adansonii (AN) can stimulate milk production in lactating women, experiments were performed to determine the effect of an aqueous extract of AN on milk production in rats. Female rats that received oral doses of aqueous extract of this plant during their first lactation produced about 59% more milk than controls (P<0.01). Pup weight gain was also significantly higher than that in the control group. A lower dose, comparable to that used by women to improve their milk yield, led to about 33% more milk with the same growth rate for pups as that in the high-dose group. The extract of AN was found to stimulate the synthesis and release of prolactin (PRL) significantly (P<0.05). In addition, the mammary glands of oestrogen-primed rats treated with the extract showed clear lobuloalveolar development with milk secretion. This study demonstrates that the aqueous extract of AN can stimulate milk production and PRL release in the female rat and could consequently have the properties claimed for lactating women.

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Photosynthesis and sink activity of wasp-induced galls in Acacia pycnantha.

Although insect galls are widely known to influence source-sink relationships in plants, the relationship between photosynthesis and gall activity has not been extensively studied. In this study we used 14CO2, photosynthesis, and respiration measurements to examine the capacity of bud galls induced by the wasp Trichilogaster signiventris (Pteromalidae) as carbon sinks in Acacia pycnantha. Galls of this species develop either in vegetative or reproductive buds, depending on the availability of tissues at different times of the year, and effectively eliminate seed production by the plant. Photosynthetic rates in phyllodes subtending clusters of galls were greater than rates in control phyllodes, a result we attributed to photosynthesis compensating for increased carbon demand by the galls. Contrary to previous studies, we found that photosynthesis within galls contributed substantially to the carbon budgets of the galls, particularly in large, mature galls, which exhibited lower specific respiration rates allowing for a net carbon gain in the light. To determine the sink capacity and competitive potential of galls, we measured the proportion of specific radioactivity in galls originating from either vegetative or reproductive buds and found no difference between them. The proportion of the total amount of phyllode-derived 14C accumulated in both clustered and solitary galls was less than that in fruits. Galls and fruits were predominantly reliant on subtending rather than on distant phyllodes for photosynthate. Solitary galls that developed in vegetative buds constituted considerably stronger sinks than galls in clusters on inflorescences where there was competition between galls or fruits for resources from the subtending phyllode. Wasps developing in solitary vegetative galls were correspondingly significantly larger than those from clustered galls. We conclude that, in the absence of inflorescence buds during summer and fall, the ability of the wasps to cause gall formation in vegetative tissues tempers intraspecific competition and substantially increases the availability of plant resources for the development of wasps in such galls.

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Nitrogen metabolism of sheep and goats consuming Acacia brevispica and Sesbania sesban.

We described the effects of two East African browses, Acacia brevispica and Sesbania sesban, on nitrogen metabolism of sheep and goats. The A. brevispica had a substantial amount of proanthocyanidins (condensed tannins); S. sesban did not. The browses were fed at three levels in combination with vetch (Vicia dasycarpa) and teff straw (Eragrostis abyssinica). Fecal N, N balance, and plasma urea N (PUN) were estimated with intact animals. Ruminal ammonia (RuA) and VFA concentrations were estimated with ruminally fistulated animals. Urinary N loss, PUN, RuA, and VFA concentrations were higher for S. sesban diets than for A. brevispica diets. Fecal N was highest with diets including A. brevispica due to high levels of fecal neutral-detergent insoluble N. Nitrogen retention was highest for diets including S. sesban. Nitrogen retention was adequate for A. brevispica diets because low urinary N compensated for high fecal N. Four hypotheses describe possible effects of tannins on N metabolism: 1) escape of protein from the rumen to the lower tract; 2) increased microbial yield; 3) increase in N-containing endogenous products; and 4) protein made indigestible in tannin-protein complexes. The effect of tannins in A. brevispica on N metabolism can best be described by the formation of indigestible tannin-protein complexes, although increased production of endogenous products is also possible.

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Preparation of microencapsulated liposomes, II. Systems containing nylon-gelatin and nylon-gelatin-acacia walling material.

Liposome suspension was encapsulated and isolated in nylon-gelatin and nylon-gelatin-acacia walled microcapsules. The resulting liposomal microcapsules could be stored in the dry state as a free-flowing powder. Liposomes remained intact after the microencapsulation, and encapsulation efficiency was greater than 90 per cent. The release of drug from microcapsules was retarded in the presence of drug-loaded liposomes.

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Influence of pH adjustment on microcapsules obtained from complex coacervation of gelatin and acacia.

The coacervation behaviour of commercial grade gelatin and acacia mixtures was studied with five different acids to adjust the coacervation pH, i.e. HCl, HNO3, H2SO4, acetic acid, and citric acid. The electrical equivalence pH value (EEP) of the polymer mixture was determined by means of a streaming current detector (SCD). With all acids--except H2SO4--maximum coacervate yield was observed at the EEP. Using H2SO4 the EEP was found at a lower pH value than compared with the point of maximum coacervate yield. The quantity of coacervate at the EEP was significantly reduced in the presence of H2SO4 whereas with all other acids, almost no differences were found. The dependence of the coacervate volume on the added amount of acid did not change in parallel to the dry coacervate yield and there was no coincidence of the maximum coacervate volume and the EEP. The barrier properties of the capsule shells of corresponding microcapsules using indomethacin as a model drug were examined by dissolution studies. Indomethacin microcapsules showed the slowest release rate when the coacervation pH was adjusted to the EEP and not to the pH of maximum coacervate yield. As expected from the coacervation behaviour, dissolution profiles of the microcapsules were quite similar even when different acids were used for pH adjustment.

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