<Emphasis Type="Italic">In vitro</Emphasis> antileishmanial activity of <Emphasis Type="Italic">Aloe vera</Emphasis> leaf exudate: A potential herbal therapy in leishmaniasis

Aloe vera has wide spread use in health products, and despite several reports on the whole plant and inner gel, little work has been performed on the leaf exudate. Our aim was to evaluate the in vitro efficacy of Aloe vera leaf exudate (AVL) in leishmaniasis. Irrespective of the disease manifestation, promastigotes from strains responsible for cutaneous, mucocutaneous, and visceral leishmaniasis were susceptible to AVL and their IC₅₀ ranged from 100 to 180 μg/ml. In axenic amastigotes cultured from a L. donovani strain 2001 responsible for visceral leishmaniasis, the IC₅₀ was 6.0 μg/ml. AVL caused activation of host macrophages evident by an increased release of members of reactive oxygen species that was attenuated by preincubation with free radical scavengers. Collectively, our data indicates that AVL, via its direct leishmanicidal activity which can be further enhanced by activation of host macrophages, is an effective antileishmanial agent meriting further pharmacological investigations.     

Cloning,characterization, and transformation of the phosphoethanolamine <Emphasis Type="Italic">N</Emphasis>-methyltransferase gene (<Emphasis Type="Italic">ZmPEAMT1</Emphasis>) in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

N-methylation of phosphoethanolamine, the committing step in choline (Cho) biosynthesis in plants, is catalyzed by S-adenosyl-l-methionine: phosphoethanolamine N-methyltransferase (PEAMT, EC 2.1.1.103). Herein we report the cloning and characterization of the novel maize phosphoethanolamine N-methyltransferase gene (ZmPEAMT1) using a combination of bioinformatics and a PCR-based allele mining strategy. The cDNA sequence of ZmPEAMT1 gene is 1,806 bp in length and translates a 495 amino acids peptide. The upstream promoter sequence of ZmPEAMT1 were obtained by TAIL-PCR, and contained four kinds of putative cis-acting regulatory elements, including stress-responsive elements, phytohormone-responsive elements, pollen developmental special activation elements, and light-induced signal transduction elements, as well as several other structural features in common with the promoter of rice and Arabidopsis homologies. RT-PCR analysis showed that expression of ZmPEAMT1 was induced by salt stress and suppressed by high temperature. Over-expression of ZmPEAMT1 enhanced the salt tolerance, root length, and silique number in transgenic Arabidopsis. These data indicated that ZmPEAMT1 maybe involved in maize root development and stress resistance, and maybe having a potential application in maize genetic engineering. Note: Nucleotide sequence data are available in GenBank under the following accession numbers: maize (Zea mays, ZmPEAMT1, AY626156; ZmPEAMT2, AY103779); rice (Oryza sativa, OsPEAMT1/Os01g50030, NM_192178; OsPEAMT2/Os05g47540, XM_475841); wheat (Triticum aestivum, TaPEAMT, AY065971); Arabidopsis (Arabidopsis thaliana, AtNMT1/At3g18000, AY091683; AtNMT2/At1g48600, NM_202264; AtNMT3/At1g73600, NM_106018); oilseed rape (Brassica napus, BnPEAMT, AY319479), tomato (Lycopersicon esculentum, AF328858), spinach (Spinacia oleracea, AF237633).     

Molecular characterization demonstrates that the <Emphasis Type="Italic">Zea mays</Emphasis> gene <Emphasis Type="Italic">sugary2</Emphasis> codes for the starch synthase isoform SSIIa

Mutations in the maize gene sugary2 (su2) affect starch structure and its resultant physiochemical properties in useful ways, although the gene has not been characterized previously at the molecular level. This study tested the hypothesis that su2 codes for starch synthase IIa (SSIIa). Two independent mutations of the su2 locus, su2-2279 and su2-5178, were identified in a Mutator-active maize population. The nucleotide sequence of the genomic locus that codes for SSIIa was compared between wild type plants and those homozygous for either novel mutation. Plants bearing su2-2279 invariably contained a Mutator transposon in exon 3 of the SSIIa gene, and su2-5178 mutants always contained a small retrotransposon-like insertion in exon 10. Six allelic su2 ^– mutations conditioned loss or reduction in abundance of the SSIIa protein detected by immunoblot. These data indicate that su2 codes for SSIIa and that deficiency in this isoform is ultimately responsible for the altered physiochemical properties of su2 ^– mutant starches. A specific starch synthase isoform among several identified in soluble endosperm extracts was absent in su2-2279 or su2-5178 mutants, indicating that SSIIa is active in the soluble phase during kernel development. The immediate structural effect of the su2 ^– mutations was shown to be increased abundance of short glucan chains in amylopectin and a proportional decrease in intermediate length chains, similar to the effects of SSII deficiency in other species.     

<Emphasis Type="Italic">N</Emphasis>-Acetyltransferase Mpr1 confers ethanol tolerance on <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> by reducing reactive oxygen species

N-Acetyltransferase Mpr1 of Saccharomyces cerevisiae can reduce intracellular oxidation levels and protect yeast cells under oxidative stress, including H₂O₂, heat-shock, or freeze-thaw treatment. Unlike many antioxidant enzyme genes induced in response to oxidative stress, the MPR1 gene seems to be constitutively expressed in yeast cells. Based on a recent report that ethanol toxicity is correlated with the production of reactive oxygen species (ROS), we examined here the role of Mpr1 under ethanol stress conditions. The null mutant of the MPR1 and MPR2 genes showed hypersensitivity to ethanol stress, and the expression of the MPR1 gene conferred stress tolerance. We also found that yeast cells exhibited increased ROS levels during exposure to ethanol stress, and that Mpr1 protects yeast cells from ethanol stress by reducing intracellular ROS levels. When the MPR1 gene was overexpressed in antioxidant enzyme-deficient mutants, increased resistance to H₂O₂ or heat shock was observed in cells lacking the CTA1, CTT1, or GPX1 gene encoding catalase A, catalase T, or glutathione peroxidase, respectively. These results suggest that Mpr1 might compensate the function of enzymes that detoxify H₂O₂. Hence, Mpr1 has promising potential for the breeding of novel ethanol-tolerant yeast strains.     

In planta transformation of <Emphasis Type="Italic">Notocactus scopa</Emphasis> cv. <Emphasis Type="Italic">Soonjung</Emphasis> by <Emphasis Type="Italic">Agrobacterium </Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>

Notocactus scopa cv. Soonjung was subjected to in planta Agrobacterium tumefaciens-mediated transformation with vacuum infiltration, pin-pricking, and a combination of the two methods. The pin-pricking combined with vacuum infiltration (20-30 cmHg for 15 min) resulted in a transformation efficiency of 67-100%, and the expression of the uidA and nptII genes was detected in transformed cactus. The established in planta transformation technique generated a transgenic cactus with higher transformation efficiency, shortened selection process, and stable gene expression via asexual reproduction. All of the results showed that the in planta transformation method utilized in the current study provided an efficient and time-saving procedure for the delivery of genes into the cactus genome, and that this technique can be applied to other asexually reproducing succulent plant species.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Improvement of <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation in Hi-II maize (<Emphasis Type="Italic">Zea mays</Emphasis>) using standard binary vectors

High-frequency transformation of maize (Zea mays L.) using standard binary vectors is advantageous for functional genomics and other genetic engineering studies. Recent advances in Agrobacterium tumefaciens-mediated transformation of maize have made it possible for the public to transform maize using standard binary vectors without a need of the superbinary vector. While maize Hi-II has been a preferred maize genotype to use in various maize transformation efforts, there is still potential and need in further improving its transformation frequency. Here we report the enhanced Agrobacterium-mediated transformation of immature zygotic embryos of maize Hi-II using standard binary vectors. This improved transformation process employs low-salt media in combined use with antioxidant l-cysteine alone or l-cysteine and dithiothreitol (DTT) during the Agrobacterium infection stage. Three levels of N6 medium salts, 10, 50, and 100%, were tested. Both 10 and 50% salts were found to enhance the T-DNA transfer in Hi-II. Addition of DTT to the cocultivation medium also improves the T-DNA transformation. About 12% overall and the highest average of 18% transformation frequencies were achieved from a large number of experiments using immature embryos grown in various seasons. The enhanced transformation protocol established here will be advantageous for maize genetic engineering studies including transformation-based functional genomics.     

Characterization of a Novel Phosphoinositide-Specific Phospholipase C from <Emphasis Type="Italic">Zea mays</Emphasis> and its Expression in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A cDNA encoding a phosphoinositide-specific phospholipase C (PI-PLC) has been isolated from Zea mays by screening a cDNA library. The cDNA, designated ZmPLC, encodes a polypeptide of 586 amino acids, containing the catalytic X, Y and C2 domains found in all PI-PLCs from plants. Northern blot analysis showed that the expression of the ZmPLC gene in roots is up-regulated under conditions of high salt, dehydration, cold or low osmotic stress conditions. Recombinant ZmPLC protein was expressed in Esch- erichia coli, purified and used to produce polyclonal antibody, this polyclonal antibody is important for further studies to assess the ultimate function of the ZmPLC gene in plants.     

Two new records of <Emphasis Type="Italic">Mycena</Emphasis> sect. <Emphasis Type="Italic">Sacchariferae</Emphasis> from Japan and type study of <Emphasis Type="Italic">Mycena cryptomeriicola</Emphasis> (sect. <Emphasis Type="Italic">Sacchariferae</Emphasis>)

Mycena cupulicola sp. nov. and M. adscendens var. carpophila, new to Japan, are described and illustrated. The former is characterized by having lageniform caulocystidia with a slightly thick-walled broadened base and no cheilocystidia. The latter is characterized by having a white pileus up to 1mm in diameter and narrowly conical caulocystidia. Mycena cryptomeriicola was confirmed to have inamyloid basoidiospores.     

<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> plants overexpressing <Emphasis Type="Italic">Ramosa1</Emphasis> maize gene show an increase in organ size due to cell expansion

The structure of the plant inflorescence and flower is an important agronomic and ornamental trait studied for its potential economic applications. In particular, the capacity to modify flower size has always been a breeder’s goal. Genetic and molecular studies have shown that the Zea mays gene Ramosa1 (Ra1) is involved in inflorescence branching regulation. In fact the ra1 loss of function mutation causes extra branching of the inflorescence. In this work we suggest a possible utilization of the Ramosa1 maize gene as a tool to modify inflorescence architecture and flower size in transgenic plants. In fact overexpression of this gene in Arabidopsis plants promotes an increase in reproductive organ size. Pollen, seeds, cotyledons, leaves and roots are also larger than those of the wild type. Analysis of organs from transformants showed that cell expansion was increased without apparently affecting cell division. These results suggest that the RA1 protein is able to up-regulate cell expansion in all organs of Arabidopsis plants.     

Floral development and systematic position of <Emphasis Type="Italic">Arillastrum</Emphasis>, <Emphasis Type="Italic">Allosyncarpia</Emphasis>, <Emphasis Type="Italic">Stockwellia</Emphasis> and <Emphasis Type="Italic">Eucalyptopsis</Emphasis> (Myrtaceae)

We have investigated the floral ontogeny of Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis (of the eucalypt group, Myrtaceae) using scanning electron microscopy and light microscopy. Several critical characters for establishing relationships between these genera and to the eucalypts have been determined. The absence of compound petaline primordia in Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis excludes these taxa from the eucalypt clade. Post-anthesis circumscissile abscission of the hypanthium above the ovary in Stockwellia, Eucalyptopsis and Allosyncarpia is evidence that these three taxa form a monophyletic group; undifferentiated perianth parts and elongated fusiform buds are characters that unite Stockwellia and Eucalyptopsis as sister taxa. No floral characters clearly associate Arillastrum with either the eucalypt clade or the clade of Stockwellia, Eucalyptopsis and Allosyncarpia.We gratefully acknowledge Clyde Dunlop and Bob Harwood (Northern Territory Herbarium) for collecting specimens of Allosyncarpia, and Bruce Gray (Atherton) for collecting specimens of Stockwellia. The Australian National Herbarium (CANB) kindly lent herbarium specimens of Eucalyptopsis for examination. This research was supported by a University of Melbourne Research Development Grant to Andrew Drinnan.     

A new species of <Emphasis Type="Italic">Gymnopilus</Emphasis> Sect. <Emphasis Type="Italic">Microspori</Emphasis> from Japan

Gymnopilus ombrophilus sp. nov., growing on rotten wood of conifers and hardwoods from Niigata, Japan is described and illustrated. It is characterized by its medium-sized, brownish-orange basidiocarps with a finely squamulose pileus, stipe lacking an annulus, and mild taste, and microscopically by dextrinoid, small, broadly ellipsoid basidiospores. The new species belongs to the section Microspori. The differences between the taxon and similar species are briefly discussed.     

<Emphasis Type="Italic">Drosophila</Emphasis><Emphasis Type="Italic">P</Emphasis> transposons of the urochordata <Emphasis Type="Italic">Ciona intestinalis</Emphasis>

P transposons belong to the eukaryotic DNA transposons, which are transposed by a cut and paste mechanism using a P-element-coded transposase. They have been detected in Drosophila, and reside as single copies and stable homologous sequences in many vertebrate species. We present the P elements Pcin1, Pcin2 and Pcin3 from Ciona intestinalis, a species of the most primitive chordates, and compare them with those from Ciona savignyi. They showed typical DNA transposon structures, namely terminal inverted repeats and target site duplications. The coding region of Pcin1 consisted of 13 small exons that could be translated into a P-transposon-homologous protein. C. intestinalis and C. savignyi displayed nearly the same phenotype. However, their P elements were highly divergent and the assumed P transposase from C. intestinalis was more closely related to the transposase from Drosophila melanogaster than to the transposase of C. savignyi. The present study showed that P elements with typical features of transposable DNA elements may be found already at the base of the chordate lineage. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

New species in <Emphasis Type="Italic">Barleria</Emphasis> sect. <Emphasis Type="Italic">Stellatohirta</Emphasis> (<Emphasis Type="Italic">Acanthaceae</Emphasis>) from Africa

Summary  Three new species are described in Barleria L. sect. Stellatohirta M. Balkwill from tropical Africa: B. aristata from south-central Tanzania, B. aenea from south-western Tanzania and northeast Zambia, and B. purpureotincta from south-western Zambia. Their affinities and conservation status are discussed.     

Life cycle of <Emphasis Type="Italic">Uromyces appendiculatus</Emphasis> var. <Emphasis Type="Italic">azukicola</Emphasis> on <Emphasis Type="Italic">Vigna angularis</Emphasis>

Field observations and inoculation experiments revealed that Uromyces appendiculatus var. azukicola has an autoecious and macrocyclic life cycle and produces spermogonia, aecia, uredinia, and telia on Vigna angularis var. angularis and V. angularis var. nipponensis. From inoculation experiments, it was suggested that this rust fungus has different host relationships from other varieties. Morphological examinations revealed that the characteristics of urediniospores and teliospores are different among varieties, although aeciospores are morphologically similar to each other.Contribution no. 182, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

<Emphasis Type="Italic">Piriformospora indica</Emphasis> and <Emphasis Type="Italic">Sebacina vermifera</Emphasis> increase growth performance at the expense of herbivore resistance in <Emphasis Type="Italic">Nicotiana attenuata</Emphasis>

A Sebacinales species was recovered from a clone library made from a pooled rhizosphere sample of Nicotiana attenuata plants from 14 native populations. Axenic cultures of the related species, Piriformospora indica and Sebacina vermifera, were used to examine their effects on plant performance. Inoculation of N. attenuata seeds with either fungus species stimulated seed germination and increased growth and stalk elongation. S. vermifera inoculated plants flowered earlier, produced more flowers and matured more seed capsules than did non-inoculated plants. Jasmonate treatment during rosette-stage growth, which slows growth and elicits herbivore resistance traits, erased differences in vegetative, but not reproductive performance resulting from S. vermifera inoculation. Total nitrogen and phosphorous contents did not differ between inoculated and control plants, suggesting that the performance benefits of fungal inoculation did not result from improvements in nutritional status. Since the expression of trypsin proteinase inhibitors (TPI), defensive proteins which confer resistance to attack from Manduca sexta larvae, incur significant growth and fitness costs for the plant, we examined the effect of S. vermifera inoculation on herbivore resistance and TPI activity. After 10 days of feeding on S. vermifera-inoculated plants, larval mass was 46% higher and TPI activity was 48% lower than that on non-inoculated plants. These results suggest that Sebacina spp. may interfere with defense signaling and allow plants to increase growth rates at the expense of herbivore resistance mediated by TPIs.