Immunomodulatory effect of exo-polysaccharides from submerged cultured <Emphasis Type="Italic">Cordyceps sinensis</Emphasis>: enhancement of cytokine synthesis,CD11b expression,and phagocytosis

Cordyceps sinensis is widely used as a traditional medicine for treatment of a wide variety of diseases or to maintain health. The immunomodulatory activity of polysaccharides prepared from submerged cultured C. sinensis BCRC36421 was investigated in human peripheral blood. Results demonstrated that Fr. A (exo-polysaccharides, 0.025 ∼ 0.1 mg/ml) induced the production of tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, and IL-10 dose-dependently. Fr. A, as low as 0.025 mg/ml, could significantly augment surface expression of CD11b in monocytes and polymorphonuclear neutrophils. Functional assay revealed that Fr. A (0.05 mg/ml) also elevated phagocytosis in monocytes and PMN. On the other hand, Fr. B (intracellular polysaccharides) only moderately induced TNF-α release, CD11b expression, and phagocytosis at the same concentrations. Our results indicate that the immunomodulatory components of submerged cultured C. sinensis mainly reside in the culture filtrate.     

Nine microsatellite DNA primers for <Emphasis Type="Italic">Hippophae rhamnoides</Emphasis> ssp. <Emphasis Type="Italic">sinensis</Emphasis> (Elaeagnaceae)

Hippophae rhamnoides ssp. sinensis occurs mainly in the arid regions of northwest China. The wood stands of this subspecies play an important role in maintaining the local ecosystems in these regions. In addition, the genetic characteristics are essential to understand the historical range changes of this subspecies and its morphological differentiation with other subspecies. In this study, we developed nine microsatellite loci for this subspecies for the first time. We used the combining biotin capture method to enrich AG/CT/AC/GT/CG/GTG/CCA microsatellites. Twenty-six microsatellites were isolated from the enriching library and nine of them were found to be polymorphic through screening 12 distantly distributed individuals. The number of alleles per locus ranged from three to twelve and expected heterozygosity from 0.2659 to 0.4767, respectively. We further performed cross-priming tests in another subspecies and two congeneric species. These firstly isolated loci will provide a useful tool to investigate the genetic structure of this subspecies and its morphological differentiation from the other subspecies.     

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.     

Isolation and characterization of polyphenol oxidase- and peroxidase-producing <Emphasis Type="Italic">Bacillus</Emphasis> strains from fully fermented tea (<Emphasis Type="Italic">Camellia sinensis</Emphasis>)

Sixteen aerobic endospore-forming Bacillus spp. were isolated from fully fermented tea leaf samples from 10 tea factories in Lahijan and Langrod cities (Gillan province, Iran). Bacillus spp. isolates were characterized using phenotypic characteristics, antibiotic susceptibility and cellular fatty acid (CFA) patterns. Based on the data obtained, five isolates of tea Bacillus spp. (TB): TB2, TB4, TB6, TB10 and TB12 belonged to the species B. subtilis. Two isolates, TB1 and TB14 were recognized as B. licheniformis. Two Bacillus spp. isolates, TB9 and TB 16 were identified as B. sphaericus. Two isolates, TB5 and TB13 were shown to be B. pumilus. Two isolates, TB7 and TB15 belonged to B. cereus. Amongst the isolates, Bacillus sp. TB3, Bacillus sp. TB8 and Bacillus sp. TB11 showed different phenotypic traits, distinct antibiotic sensitivity and fatty acid profiles, and they may represent novel species. The isolates showed polyphenol oxidase (tyrosinase) and peroxidase activities. The highest polyphenol oxidase and peroxidase activities were observed for Bacillus sp. TB3 and B. licheniformis TB14, respectively, where values of 5.48 and 3.73 units mL⁻¹ were observed.     

Cloning and expression of selenocysteine methyltransferase cDNA from <Emphasis Type="Italic">Camellia sinensis</Emphasis>

Selenocysteine methyltransferase (SMT), specifically methylates selenocysteine (SeCys) to produce the nonprotein amino acid Se-methyl selenocysteine (SeMSC) and played key role of removing selenium toxic effect at higher levels to the plant. Here we report the cloning of a cDNA encoding selenocysteine methyltransferase from Camellia sinensis (CsSMT) and expression of CsSMT in Escherichia coli. CsSMT isolated by RT-PCR and RACE-PCR reaction. CsSMT is a 1,401 bp cDNA with an open reading frame predicted to encode a 351 amino acid, 40.5 kDa protein; The predicted amino acid sequences of CsSMT shows 74% identity with A. bisulcatus selenocysteine methyltransferase (AbSMT) and 69% identity with Broccoli (Brassica oleracea var. italica) selenocysteine methyltransferase (BoSMT), and shares 53, 73 and 65% identity, respectively, with Arabidopsis thaliana homocysteine S-methyltransferase AtHMT1, AtHMT2, and AtHMT3, and 65% to Zea mays homocysteine S-methyltransferase (ZmHMT2). Analyses of CsSMT showed that it lacks obvious chloroplast or mitochondrial targeting sequences and contains a consensus sequence of GGCC for a possible zinc-binding motif near the C-terminal and a conserved Cys residue upstream of the zinc-binding motif as other related methyltransferases. Expression of CsSMT correlated with the presence of SMT enzyme activity in cell extracts, and bacteria containing recombinant CsSMT plasmid showed much high tolerance to selenate and selenite.     

Asymmetric somatic hybridization between UV-irradiated <Emphasis Type="Italic">Citrus unshiu</Emphasis> and <Emphasis Type="Italic">C</Emphasis>. <Emphasis Type="Italic">sinensis</Emphasis>: regeneration and characterization of hybrid shoots

In the present paper, attempts were made to explore the possibility of employing ultraviolet (UV) irradiation in citrus asymmetric fusion for transfer of limited amount of favorable traits from a desirable cultivar to a target one. Exposure of Satsuma mandarin (Citrus unshiu Marc.) embryogenic protoplasts to UV at an intensity of 300 μW cm⁻² led to reduced viability, especially under long irradiation duration. The protoplasts could not grow during culture when they were irradiated for over 30 s. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay revealed extensive DNA fragmentation in the UV-irradiated protoplasts compared with those without UV treatment. Electrofusion between UV-irradiated protoplasts of Satsuma mandarin (donor) with those of Jincheng (C. sinensis Osbeck, recipient), a local cultivar of superior quality, gave rise to regeneration of several lines of shoots, which failed to root despite enormous endeavors. Ploidy analysis via flow cytometry and chromosome counting showed that four selected shoots were either diploid, triploid or tetraploid. Random amplified polymorphism DNA (RAPD) and amplified fragment length polymorphism (AFLP) confirmed the shoots, irrespective of their ploidy level, as putative somatic hybrids. Cleaved amplified polymorphism sequences (CAPS) demonstrated that the shoots predominantly got their cytoplasmic components, in terms of chloroplast (cp) and mitochondrion DNA, from Jincheng, along with possible recombination of cpDNA in some shoot lines. The current data indicated that UV-based asymmetric fusion could also be employed in citrus somatic hybridization with the intention of creating novel germplasms, which may provide an alternative approach for cultivar improvement.     

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.     

<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.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-Mediated genetic transformation in tea (<Emphasis Type="Italic">Camellia sinensis</Emphasis> [L.] O. Kuntze)

We have developed a system to produce transgenic plants in tea (Camelia sinensis [L.] O. Kuntze) viaAgrobacterium tumefaciens-mediated transformation of embryogenic calli. Cotyledon-derived embryogenic callus cultures were cocultivated with anA. tumefaciens strain (AGL 1) harboring a binary vector carrying the hygromycin phosphotransferase (hpt II), glucuronidase (uid A), and green fluorescent protein (GFP) genes in the tDNA region. Following cocultivation, embryogenic calli were cultured in medium containing 500 mg/L carbenicillin for 1 wk and cultured on an antibiotic selection medium containing 75 mg/L hygromycin for 8–10 wk. Hygromycin-resistant somatic embryos were selected. The highest production efficiency of hygromycin-resistant calli occurred with cocultivation for 6–7 d in the presence of 400 μM acetosyringone (AS). Hygromycin-resistant somatic embryos developed into complete plantlets in regeneration medium containing half-strength Murashige and Skoog (MS) salts with 1 mg/L benzyl amino purine (BAP) and 9 mg/L giberellic acid (GA₃). Transformants were subjected to GFP expression analysis, β-glucuronidase (GUS) histochemical assay, PCR analysis, and Southern hybridization to confirm gene integration.     

<Emphasis Type="Italic">Funastrum rupicola</Emphasis> (<Emphasis Type="Italic">Apocynaceae:</Emphasis><Emphasis Type="Italic">Asclepiadoideae</Emphasis>), a new species from Bolivia

Summary   Funastrum rupicola Goyder, a new species of Apocynaceae: Asclepiadoideae from Bolivia, is described and illustrated. The conservation status of this species is assessed.     

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.     

Phylogenetic analyses of <Emphasis Type="Italic">Uromyces viciae-fabae</Emphasis> and its varieties on <Emphasis Type="Italic">Vicia</Emphasis>, <Emphasis Type="Italic">Lathyrus</Emphasis>, and <Emphasis Type="Italic">Pisum</Emphasis> in Japan

A pea rust fungus, Uromyces viciae-fabae, has been classified into two varieties, var. viciae-fabae and var. orobi, based on differences in urediniospore wall thickness and putative host specificity in Japan. In principal component analyses, morphological features of urediniospores and teliospores of 94 rust specimens from Vicia, Lathyrus, and Pisum did not show definite host-specific morphological groups. In molecular analyses, 23 Uromyces specimens from Vicia, Lathyrus, and Pisum formed a single genetic clade based on D1/D2 and ITS regions. Four isolates of U. viciae-fabae from V. cracca and V. unijuga could infect and sporulate on P. sativum. These results suggest that U. viciae-fabae populations on different host plants are not biologically differentiated into groups that can be recognized as varieties.Contribution no. 184, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

<Emphasis Type="Italic">Phuphanochloa,</Emphasis> a new bamboo genus (<Emphasis Type="Italic">Poaceae</Emphasis>: <Emphasis Type="Italic">Bambusoideae</Emphasis>) from Thailand

Summary  A new monotypic bamboo genus Phuphanochloa (Poaceae: Bambusoideae) from north-eastern Thailand is described, together with a new species, P. speciosa.     

Regulation of stipule development by <Emphasis Type="Italic">COCHLEATA</Emphasis> and <Emphasis Type="Italic">STIPULE</Emphasis>-<Emphasis Type="Italic">REDUCED</Emphasis> genes in pea <Emphasis Type="Italic">Pisum sativum</Emphasis>

Pisum sativum L., the garden pea crop plant, is serving as the unique model for genetic analyses of morphogenetic development of stipule, the lateral organ formed on either side of the junction of leafblade petiole and stem at nodes. The stipule reduced (st) and cochleata (coch) stipule mutations and afila (af), tendril-less (tl), multifoliate-pinna (mfp) and unifoliata-tendrilled acacia (uni-tac) leafblade mutations were variously combined and the recombinant genotypes were quantitatively phenotyped for stipule morphology at both vegetative and reproductive nodes. The observations suggest a role of master regulator to COCH in stipule development. COCH is essential for initiation, growth and development of stipule, represses the UNI-TAC, AF, TL and MFP led leafblade-like morphogenetic pathway for compound stipule and together with ST mediates the developmental pathway for peltate-shaped simple wild-type stipule. It is also shown that stipule is an autonomous lateral organ, like a leafblade and secondary inflorescence.     

Study of tauopathies by comparing <Emphasis Type="Italic">Drosophila</Emphasis> and human <Emphasis Type="Italic">tau</Emphasis> in <Emphasis Type="Italic">Drosophila</Emphasis>

The microtubule-binding protein tau has been investigated for its contribution to various neurodegenerative disorders. However, the findings from transgenic studies, using the same tau transgene, vary widely among different laboratories. Here, we have investigated the potential mechanisms underlying tauopathies by comparing Drosophila (d-tau) and human (h-tau) tau in a Drosophila model. Overexpression of a single copy of either tau isoform in the retina results in a similar rough eye phenotype. However, co-expression of Par-1 with d-tau leads to lethality, whereas co-expression of Par-1 with h-tau has little effect on the rough eye phenotype. We have found analogous results by comparing larval proteomes. Through genetic screening and proteomic analysis, we have identified some important potential modifiers and tau-associated proteins. These results suggest that the two tau genes differ significantly. This comparison between species-specific isoforms may help to clarify whether the homologous tau genes are conserved. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. This study was supported by the National Science Foundation of China (30270341; 30630028), the Multidisciplinary Program (Brain and Mind) of the Chinese Academy of Sciences, the Major State Basic Research Program (“973 program”; G2000077800; G2006CB806600; 2006CB911003), the Precedent Project of Important Intersectional Disciplines in the Knowledge Innovation Engineering of the Chinese Academy of Sciences (KJCX1-09-03).     

<Emphasis Type="Italic">In vitro</Emphasis> culture of the resurrection plant <Emphasis Type="Italic">Haberlea rhodopensis</Emphasis>

The small group of resurrection plants is a unique model which could help us in further understanding of abiotic stress tolerance. The most frequently used approach for investigations on gene functions in plant systems is genetic transformation. In this respect, the establishment of in vitro systems for regeneration and micro propagation is necessary. On the other hand, in vitro cultures of such rare plants could preserve their natural populations. Here, we present our procedure for in vitro regeneration and propagation of Haberlea rhodopensis – a resurrection plant species, endemic for the Balkan region.     

<Emphasis Type="Italic">Typhonium stigmatilobatum</Emphasis> (<Emphasis Type="Italic">Araceae</Emphasis> tribe <Emphasis Type="Italic">Areae</Emphasis>), a new species from Vietnam

Summary   Typhonium stigmatilobatum V. D. Nguyen, a new species from Vietnam, is described and illustrated.