An <Emphasis Type="SmallCaps">l</Emphasis>-Glutamine Transporter Isoform for Neurogenesis Facilitated by <Emphasis Type="SmallCaps">l</Emphasis>-Theanine

l-Theanine (=γ-glutamylethylamide) is an amino acid ingredient in green tea with a structural analogy to l-glutamine (l-GLN) rather than l-glutamic acid (l-GLU), with regards to the absence of a free carboxylic acid moiety from the gamma carbon position. l-theanine markedly inhibits [³H]l-GLN uptake without affecting [³H]l-GLU uptake in cultured neurons and astroglia. In neural progenitor cells with sustained exposure to l-theanine, upregulation of the l-GLN transporter isoform Slc38a1 expression and promotion of both proliferation and neuronal commitment are seen along with marked acceleration of the phosphorylation of mammalian target of rapamycin (mTOR) and relevant downstream proteins. Stable overexpression of Slc38a1 leads to promotion of cellular growth with facilitated neuronal commitment in pluripotent embryonic carcinoma P19 cells. In P19 cells stably overexpressing Slc38a1, marked phosphorylation is seen with mTOR and downstream proteins in a fashion insensitive to the additional stimulation by l-theanine. The green tea amino acid l-theanine could thus elicit pharmacological actions to up-regulate Slc38a1 expression for activation of the mTOR signaling pathway required for cell growth together with accelerated neurogenesis after sustained exposure in undifferentiated neural progenitor cells. In this review, I summarize a novel pharmacological property of the green tea amino acid l-theanine for embryonic and adult neurogenesis with a focus on the endogenous amino acid analog l-GLN. A possible translational strategy is also discussed on the development of dietary supplements and nutraceuticals enriched of l-theanine for the prophylaxis of a variety of untoward impairments and malfunctions seen in patients with different neurodegenerative and/or neuropsychiatric disorders.     

Synthesis of theanine from glutamic acid <Emphasis Type="Italic">γ</Emphasis>-methyl ester and ethylamine catalyzed by <Emphasis Type="Italic">Escherichia coli</Emphasis> having <Emphasis Type="Italic">γ</Emphasis>-glutamyltranspeptidase activity

Glutamic acid γ-methyl ester (GAME) was used as substrate for theanine synthesis catalyzed by Escherichia coli cells possessing γ-glutamyltranspeptidase activity. The yield was about 1.2-fold higher than with glutamine as substrate. The reaction was optimal at pH 10 and 45°C, and the optimal substrate ratio of GAME to ethylamine was 1:10 (mol/mol). With GAME at 100 mmol, 95 mmol theanine was obtained after 8 h.     

In Vitro Functional Characterisation of Cytochrome P450 (CYP) 2C19 Allelic Variants <Emphasis Type="Italic">CYP2C19*23</Emphasis> and <Emphasis Type="Italic">CYP2C19*24</Emphasis>

Cytochrome P450 (CYP) 2C19 is essential for the metabolism of clinically used drugs including omeprazole, proguanil, and S-mephenytoin. This hepatic enzyme exhibits genetic polymorphism with inter-individual variability in catalytic activity. This study aimed to characterise the functional consequences of CYP2C19*23 (271 G>C, 991 A>G) and CYP2C19*24 (991 A>G, 1004 G>A) in vitro. Mutations in CYP2C19 cDNA were introduced by site-directed mutagenesis, and the CYP2C19 wild type (WT) as well as variants proteins were subsequently expressed using Escherichia coli cells. Catalytic activities of CYP2C19 WT and those of variants were determined by high performance liquid chromatography-based essay employing S-mephenytoin and omeprazole as probe substrates. Results showed that the level of S-mephenytoin 4′-hydroxylation activity of CYP2C19*23 (V _max 111.5 ± 16.0 pmol/min/mg, K _m 158.3 ± 88.0 μM) protein relative to CYP2C19 WT (V _max 101.6 + 12.4 pmol/min/mg, K _m 123.0 ± 19.2 μM) protein had no significant difference. In contrast, the K _m of CYP2C19*24 (270.1 ± 57.2 μM) increased significantly as compared to CYP2C19 WT (123.0 ± 19.2 μM) and V _max of CYP2C19*24 (23.6 ± 2.6 pmol/min/mg) protein was significantly lower than that of the WT protein (101.6 ± 12.4 pmol/min/mg). In vitro intrinsic clearance (CL_int = V _max/K _m) for CYP2C19*23 protein was 85.4 % of that of CYP2C19 WT protein. The corresponding CL_int value for CYP2C19*24 protein reduced to 11.0 % of that of WT protein. These findings suggested that catalytic activity of CYP2C19 was not affected by the corresponding amino acid substitutions in CYP2C19*23 protein; and the reverse was true for CYP2C19*24 protein. When omeprazole was employed as the substrate, K _m of CYP2C19*23 (1911 ± 244.73 μM) was at least 100 times higher than that of CYP2C19 WT (18.37 ± 1.64 μM) and V _max of CYP2C19*23 (3.87 ± 0.74 pmol/min/mg) dropped to 13.4 % of the CYP2C19 WT (28.84 ± 0.61 pmol/min/mg) level. Derived from V _max/K _m, the CL_int value of CYP2C19 WT was 785 folds of CYP2C19*23. K _m and V _max values could not be determined for CYP2C19*24 due to its low catalytic activity towards omeprazole 5′-hydroxylation. Therefore, both CYP2C19*23 and CYP2C19*24 showed marked reduced activities of metabolising omeprazole to 5-hydroxyomeprazole. Hence, carriers of CYP2C19*23 and CYP2C19*24 allele are potentially poor metabolisers of CYP2C19-mediated substrates.     

<Emphasis Type="Italic">Lactobacillus paracasei</Emphasis> strain 06TCa19 suppresses inflammatory chemokine induced by <Emphasis Type="Italic">Helicobacter pylori</Emphasis> in human gastric epithelial cells

Helicobacter (H.) pylori infection is an important risk factor for gastric cancer that causes gastric inflammation. Inflammatory chemokines such as interleukin (IL)-8 and regulated on activation normal T cell expressed and secreted (RANTES) are elevated in the gastric mucosa by H. pylori. This study aimed to investigate the effects of Lactobacillus paracasei strain 06TCa19, a probiotic strain, on IL-8 and RANTES expression and production induced by H. pylori using human gastric epithelial cell lines. Strain 06TCa19 was shown to suppress H. pylori-mediated elevation of gene expression related to these chemokines in MKN45 cells. The strain also suppressed the increase in IL-8 and RANTES products induced by H. pylori in AGS cells as well as in MKN45 cells. In MKN45 cells inoculated with H. pylori, strain 06TCa19 was shown to downregulate the activation of NF-κB and p38 MAPK signaling pathways. Additionally, the level of the CagA virulence protein of H. pylori in the MKN45 cells and the number of viable H. pylori adhering to MKN45 cells decreased with the addition of strain 06TCa19. Moreover, the strain 06TCa19 notably increased lactic acid in the supernatant of MKN45 cells. Thus, lactic acid released from strain 06TCa19 might have inhibited the adhesion of H. pylori to MKN45 cells and prevented the insertion of H. pylori CagA into the cells, and elevation of IL-8 and RANTES genes and proteins might be suppressed by downregulating the NF-κB and p38 MAPK pathways. Therefore, use of strain 06TCa19 may prevent H. pylori-associated gastric inflammation.     

Evaluation of lytic bacteriophages for control of multidrug-resistant <Emphasis Type="Italic">Salmonella</Emphasis> Typhimurium

Background

The emergence of antibiotic-resistant bacteria can cause serious clinical and public health problems. This study describes the possibility of using bacteriophages as an alternative agent to control multidrug-resistant Salmonella Typhimurium.

Methods

The potential lytic bacteriophages (P22-B1, P22, PBST10, PBST13, PBST32, and PBST 35) were characterized by morphological property, heat and pH stability, optimum multiplicity of infection (MOI), and lytic activity against S. Typhimurium KCCM 40253, S. Typhimurium ATCC 19585, ciprofloxacin-induced antibiotic-resistant S. Typhimurium ATCC 19585, and S. Typhimurium CCARM 8009.

Results

P22-B1 and P22 belong to Podoviridae family and PBST10, PBST13, PBST32, and PBST 35 show a typical structure with polyhedral head and long tail, belonging to Siphoviridae family. Salmonella bacteriophages were highly stable at the temperatures (< 60 °C) and pHs (5.0–11.0). The reduction rates of host cells were increased at the MOI-dependent manner, showing the highest reduction rate at MOI of 10. The host cells were most effectively reduced by P22, while P22-B1 showed the least lytic activity. The ciprofloxacin-induced antibiotic-resistant S. Typhimurium ATCC 19585, and clinically isolated antibiotic-resistant S. Typhimurium CCARM 8009 were resistant to ciprofloxacin, levofloxacin, norfloxacin, and tetracycline. P22 showed the highest lytic activity against S. Typhimurium KCCM 40253 (> 5 log reduction), followed by S. Typhimurium ATCC 19585 (4 log reduction) and ciprofloxacin-induced antibiotic-resistant S. Typhimurium ATCC 19585 (4 log reduction).

Conclusion

The results would provide vital insights into the application of lytic bacteriophages as an alternative therapeutics for the control of multidrug-resistant pathogens.

     

Analysis of the effect of plant growth regulators and organic elicitors on antibacterial activity of <Emphasis Type="Italic">Eucomis autumnalis</Emphasis> and <Emphasis Type="Italic">Drimia robusta</Emphasis> ex vitro-grown biomass

The effects of plant growth regulators (PGRs) and organic elicitors (OEs) on in vitro propagation of Eucomis autumnalis was established. Three-year-old ex vitro grown plants from organogenesis of E. autumnalis and somatic embryogenesis (previously reported protocol) of Drimia robusta were investigated for antibacterial activity. In vitro propagation from leaf explants of E. autumnalis was established using different PGRs and OE treatments for mass propagation, biomass production and bioactivity analysis to supplement the use of wild plant material. Prolific shoots (16.0?±?0.94 shoots per explant) were obtained with MS (Murashige and Skoog in Physiol Plant 15:473–497, 1962) medium containing 100 mg l^?1 haemoglobin (HB), 10 µM benzyladenine (BA) and 2 µM naphthaleneacetic acid (NAA). The shoots were rooted effectively with a combination of 2.5 µM indole-3-acetic acid and 5.0 µM indole-3-butyric acid. The plantlets were successfully acclimatized in a vermiculite-soil mixture (1:1 v/v) in the greenhouse. Three-year-old ex vitro-grown E. autumnalis and D. robusta plants derived via organogenesis and somatic embryogenesis respectively exhibited antibacterial activity and varied with PGR and OE treatments, plant parts and bacteria. The leaves of E. autumnalis ex vitro-derived from a combination of HB, BA and NAA followed by the individual treatments of BA and HB gave the best antibacterial activities (<?1 mg ml^?1: minimum inhibitory concentration from 0.098 to 0.78 mg ml^?1) against all tested pathogenic bacteria (Bacillus subtilis, Enterococcus faecalis, Micrococcus luteus, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa). The bulbs of D. robusta ex vitro-derived from solid culture with 10 µM picloram, 1 µM thidiazuron and 20 µM glutamine exhibited good antibacterial activity against E. faecalis, M. luteus and S. aureus when compared with other treatments and mother plants. The ex vitro-grown E. autumnalis and D. robusta biomass produced with PGRs along with OE treatments confirmed a good potent bioresource and can be used as antibacterial agents. The in vitro plant regeneration of E. autumnalis and D. robusta protocols and ex vitro plants could be used for conservation strategies, bioactivity and traditional medicinal use.     

Growth and biochemical analysis of evergreen haloxeric tree species <Emphasis Type="Italic">Salvadora oleoides</Emphasis> and <Emphasis Type="Italic">Salvadora persica</Emphasis> under NaCl stress

In vitro growth, development, total soluble proteins and peroxidase profiles of Salvadora oleoides and Salvadora persica under NaCl stress were analysed in the present investigation. The plants are evergreen haloxeric tree species of family Salvadoraceae. Shoot apex from natural plants were initially used for screening of NaCl tolerance on MS culture medium. Shoot apex of S. oleoides and S. persica could survive optimally up to 200 and 100 mM NaCl. Axillary buds from nodal shoot segments of S. oleoides and S. persica were activated on 6 and 4 μM BAP, and were used further for extraction of total soluble proteins and peroxidases. Total soluble proteins were increased up to 150 mM NaCl in S. oleoides, but decline above 50 mM NaCl in S. persica. Peroxidase activity remained almost constant in S. oleoides at all the concentrations and duration of NaCl, but increased at 100 mM NaCl during fourth week of treatment in S. persica. Eleven peroxidase isozymes were observed in zymogram of S. oleoides. Isozymes P1, P2, P3, and P4 were slightly appeared, but P6 isozyme was lacking in S. persica. The P5 isozyme was more prominent in S. persica than S. oleoides. Isozyme P9 of S. persica was visible during the first week of NaCl treatment, but disappeared in the fourth week. Molecular biology of these plants can be useful further for the understanding of stress tolerance mechanisms for prospects.     

Acaricidal active fractions from acetone extract of <Emphasis Type="Italic">Aloe vera</Emphasis> L. against <Emphasis Type="Italic">Tetranychus cinnabarinus</Emphasis> and <Emphasis Type="Italic">Panonychus citri</Emphasis>

This study aimed to isolate acaricidal active fractions from acetone extract of Aloe vera L. and investigate the toxicity of these fractions against Tetranychus cinnabarinus (T. cinnabarinus) and Panonychus citri (P. citri). Acetone extract of A. vera L. was isolated by immersing in acetone for 72 h, and diverse fractions were fractionated by column chromatography. The acaricidal activity of each fractions was evaluated by corrected mortality of T. cinnabarinus through slide-dip bioassay. The 8th and 13th fractions of acetone extract with good acaricidal activity were indentified by LC/MS, and the toxicity of these two fractions to T. cinnabarinus and P. citri was identified by regression analysis. Acetone extract of A. vera L. exhibited obvious acaricidal activity, from which a total of 18 fractions were isolated. The 8th and 13th fractions with strong acaricidal activity against T. cinnabarinus were identified to be 3-O-alpha-d-mannopyranosyl-d-mannopyranose (OAMM) and aloe emodin. When compared with spirodiclofen, both OAMM and aloe emodin exhibited higher toxicity to T. cinnabarinus, while only OAMM exhibited a higher toxicity to P. citri (P < 0.05). OAMM and aloe emodin isolated from acetone extract of A. vera L. exhibited obvious acaricidal activities against T. cinnabarinus and P. citri.     

<Emphasis Type="Italic">Pseudomonas</Emphasis> spp. increases root biomass and tropane alkaloid yields in transgenic hairy roots of <Emphasis Type="Italic">Datura</Emphasis> spp.

Transgenic hairy roots of Datura spp., established using strain A4 of Agrobacterium rhizogenes, are genetically stable and produce high levels of tropane alkaloids. To increase biomass and tropane alkaloid content of this plant tissue, four Pseudomonas strains, Pseudomonas fluorescens P64, P66, C7R12, and Pseudomonas putida PP01 were assayed as biotic elicitors on transgenic hairy roots of Datura stramonium, Datura tatula, and Datura innoxia. Alkaloids were extracted from dried biomass, and hyoscyamine and scopolamine were quantified using liquid chromatography-tandem mass spectrometry analysis. D. stramonium and D. innoxia biomass production was stimulated by all Pseudomonas spp. strains after a 5-d treatment. All strains of P. fluorescens increased hyoscyamine yields compared to untreated cultures after both 5 and 10 d of treatment. Hyoscyamine yields were highest in D. tatula cultures exposed to a 5-d treatment with C7R12 (16.633 + 0.456 mg g^?1 dry weight, a 431% increase) although the highest yield increases compared to the control were observed in D. stramonium cultures exposed to strains P64 (511% increase) and C7R12 (583% increase) for 10 d. D. innoxia showed the highest scopolamine yields after elicitation with P. fluorescens strains P64 for 5 d (0.653 + 0.021 mg g^?1 dry weight, a 265% increase) and P66 for 5 and 10 d (5 d, 0.754 + 0.0.031 mg g^?1 dry weight, a 321% increase; 10 d 0.634 + 0.046 mg g^?1 dry weight, a 277% increase). These results show that the Pseudomonas strains studied here can positively and significantly affect biomass and the yields of hyoscyamine and scopolamine from transgenic roots of the three Datura species.     

Diversity and enzyme activity of <Emphasis Type="Italic">Penicillium</Emphasis> species associated with macroalgae in Jeju Island

A total of 28 strains of 19 Penicillium species were isolated in a survey of extracellular enzyme-producing fungi from macroalgae along the coast of Jeju Island of Korea. Penicillium species were identified based on morphological and β-tubulin sequence analyses. In addition, the halo-tolerance and enzyme activity of all strains were evaluated. The diversity of Penicillium strains isolated from brown algae was higher than the diversity of strains isolated from green and red algae. The commonly isolated species were Penicillium antarcticum, P. bialowiezense, P. brevicompactum, P. crustosum, P. oxalicum, P. rubens, P. sumatrense, and P. terrigenum. While many strains showed endoglucanase, β-glucosidase, and protease activity, no alginase activity was detected. There was a positive correlation between halo-tolerance and endoglucanase activity within Penicillium species. Among 19 Penicillium species, three species–P. kongii, P. olsonii, and P. viticola–have not been previously recorded in Korea.     

Production of <Emphasis Type="Italic">trans</Emphasis>-10,<Emphasis Type="Italic">cis</Emphasis>-12-conjugated linoleic acid using permeabilized whole-cell biocatalyst of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

Objective

To improve the production of trans-10,cis-12-conjugated linoleic acid (t10,c12-CLA) from linoleic acid in recombinant Yarrowia lipolytica.

Results

Cells of the yeast were permeabilized by freeze/thawing. The optimal conditions for t10,c12-CLA production by the permeabilized cells were at 28 °C, pH 7, 200 rpm with 1.5 g sodium acetate l^?1, 100 g wet cells l^?1, and 25 g LA l^?1. Under these conditions, the permeabilized cells produced 15.6 g t10,c12-CLA l^?1 after 40 h, with a conversion yield of 62 %. The permeabilized cells could be used repeatedly for three cycles, with the t10,c12-CLA extracellular production remaining above 10 g l^?1.

Conclusion

Synthesis of t10,c12-CLA was achieved using a novel method, and the production reported in this work is the highest value reported to date.

     

Improving methionine and ATP availability by <Emphasis Type="Italic">MET6</Emphasis> and <Emphasis Type="Italic">SAM2</Emphasis> co-expression combined with sodium citrate feeding enhanced SAM accumulation in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

S-adenosyl-l-methionine (SAM), biosynthesized from methionine and ATP, exhibited diverse pharmaceutical applications. To enhance SAM accumulation in S. cerevisiae CGMCC 2842 (wild type), improvement of methionine and ATP availability through MET6 and SAM2 co-expression combined with sodium citrate feeding was investigated here. Feeding 6 g/L methionine at 12 h into medium was found to increase SAM accumulation by 38 % in wild type strain. Based on this result, MET6, encoding methionine synthase, was overexpressed, which caused a 59 % increase of SAM. To redirect intracellular methionine into SAM, MET6 and SAM2 (encoding methionine adenosyltransferase) were co-expressed to obtain the recombinant strain YGSPM in which the SAM accumulation was 2.34-fold of wild type strain. The data obtained showed that co-expression of MET6 and SAM2 improved intracellular methionine availability and redirected the methionine to SAM biosynthesis. To elevate intracellular ATP levels, 6 g/L sodium citrate, used as an auxiliary energy substrate, was fed into the batch fermentation medium, and an additional 19 % increase of SAM was observed after sodium citrate addition. Meanwhile, it was found that addition of sodium citrate improved the isocitrate dehydrogenase activity which was associated with the intracellular ATP levels. The results demonstrated that addition of sodium citrate improved intracellular ATP levels which promoted conversion of methionine into SAM. This study presented a feasible approach with considerable potential for developing highly SAM-productive strains based on improving methionine and ATP availability.     

Effects of IP<Subscript>3</Subscript>R2 Receptor Deletion in the Ischemic Mouse Retina

Glial cells in the diseased nervous system undergo a process known as reactive gliosis. Gliosis of retinal Müller glial cells is characterized by an upregulation of glial fibrillary acidic protein and frequently by a reduction of inward K⁺ current amplitudes. Purinergic signaling is assumed to be involved in gliotic processes. As previously shown, lack of the nucleotide receptor P2Y₁ leads to an altered regulation of K⁺ currents in Müller cells of the ischemic retina. Here, we asked first whether this effect is mediated by the IP₃ receptor subtype 2 (IP₃R2) known as the major downstream signaling target of P2Y₁ in Müller cells. The second question was whether lack of IP₃R2 affects neuronal survival in the control and ischemic retina. Ischemia was induced in wild type and IP₃R2-deficient (IP ₃ R2 ^?/?) mice by transient elevation of the intraocular pressure. Immunostaining and TUNEL labelling were used to quantify neuronal cell loss. The downregulation of inward K⁺ currents in Müller cells from ischemic IP ₃ R2 ^?/? retinae was less strong than in wild type animals. The reduction of the number of cells in the ganglion cell layer and of calretinin- and calbindin-positive cells 7 days after ischemia was similar in wild type and IP ₃ R2 ^?/? mice. However, IP₃R2 deficiency led to an increased number of TUNEL-positive cells in the outer nuclear layer at 1 day and to an enhanced postischemic loss of photoreceptors 7 days after ischemia. This implies that IP₃R2 is involved in some but not all aspects of signaling in Müller cells after an ischemic insult.     

Molecular Cloning,Expression and Characterization of Pectin Methylesterase (<Emphasis Type="Italic">Ct</Emphasis>PME) from <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

Many phytopathogenic micro-organisms such as bacteria and fungi produce pectin methylesterases (PME) during plant invasion. Plants and insects also produce PME to degrade plant cell wall. In the present study, a thermostable pectin methylesterase (CtPME) from Clostridium thermocellum belonging to family 8 carbohydrate esterase (CE8) was cloned, expressed and purified. The amino acid sequence of CtPME exhibited similarity with pectin methylesterase from Erwinia chrysanthemi with 38% identity. The gene encoding CtPME was cloned into pET28a(+) vector and expressed using Escherichia coli BL21(DE3) cells. The recombinant CtPME expressed as a soluble protein and exhibited a single band of molecular mass approximately 35.2 kDa on SDS-PAGE gels. The molecular mass, 35.5 kDa of the enzyme, was also confirmed by MALDI-TOF MS analysis. Notably, highest protein concentration (11.4 mg/mL) of CtPME was achieved in auto-induction medium, as compared with LB medium (1.5 mg/mL). CtPME showed maximum activity (18.1 U/mg) against citrus pectin with >85% methyl esterification. The optimum pH and temperature for activity of CtPME were 8.5 and 50 °C, respectively. The enzyme was stable in pH range 8.0–9.0 and thermostable between 45 and 70 °C. CtPME activity was increased by 40% by 5 mM Ca²⁺ or Mg²⁺ ions. Protein melting curve of CtPME gave a peak at 80 °C. The peak was shifted to 85 °C in the presence of 5 mM Ca²⁺ ions, and the addition of 5 mM EDTA shifted back the melting peak to 80 °C. CtPME can be potentially used in food and textile industry applications.     

Single point mutations enhance activity of <Emphasis Type="Italic">cis</Emphasis>-epoxysuccinate hydrolase

Objectives

To enhance activity of cis-epoxysuccinate hydrolase from Klebsiella sp. BK-58 for converting cis-epoxysuccinate to tartrate.

Results

By semi-saturation mutagenesis, all the mutants of the six important conserved residues almost completely lost activity. Then random mutation by error-prone PCR and high throughput screening were further performed to screen higher activity enzyme. We obtained a positive mutant F10D after screening 6000 mutations. Saturation mutagenesis on residues Phe10 showed that most of mutants exhibited higher activity than the wild-type, and the highest mutant was F10Q with activity of 812 U mg^?1 (k _cat /K _m , 9.8 ± 0.1 mM^?1 s^?1), which was 230 % higher than that of wild-type enzyme 355 U mg^?1 (k _cat /K _m , 5.3 ± 0.1 mM^?1 s^?1). However, the thermostability of the mutant F10Q slightly decreased.

Conclusions

The catalytic activity of a cis-epoxysuccinate hydrolase was efficient improved by a single mutation F10Q and Phe10 might play an important role in the catalysis.

     

Diversity,molecular phylogeny and fingerprint profiles of airborne <Emphasis Type="Italic">Aspergillus</Emphasis> species using random amplified polymorphic DNA

In the present study, diversity and phylogenetic relationship of Aspergillus species isolated from Tehran air was studied using random amplified polymorphic DNA (RAPD)–polymerase chain reaction (RAPD-PCR). Thirty-eight Aspergillus isolates belonging to 12 species i.e. A. niger (28.94 %, 11 isolates), A. flavus (18.42 %, 7 isolates), A. tubingensis (13.15 %, 5 isolates), A. japonicus (10.52 %, 4 isolates), A. ochraceus (10.52 %, 4 isolates), and 2.63 %, 1 isolate from each A. nidulans, A. amstelodami, A. oryzae, A. terreus, A. versicolor, A. flavipes and A. fumigatus were obtained by settle plate method which they were distributed in 18 out of 22 sampling sites examined. Fungal DNA was extracted from cultured mycelia of all Aspergillus isolates on Sabouraud Dextrose Agar and used for amplification of gene fragments in RAPD-PCR using 11 primers. RAPD-PCR data was analyzed using UPGMA software. Resulting dendrogram of combined selected primers including PM1, OPW-04, OPW-05, P160, P54, P10 and OPA14 indicated the distribution of 12 Aspergillus species in 8 major clusters. The similarity coefficient of all 38 Aspergillus isolates ranged from 0.02 to 0.40 indicating a wide degree of similarities and differences within and between species. Taken together, our results showed that various Aspergillus species including some important human pathogenic ones exist in the outdoor air of Tehran by different extents in distribution and diversity and suggested inter- and intra-species genetic diversity among Aspergillus species by RAPD-PCR as a rapid, sensitive and reproducible method.     

Genomic clues to the parental origin of the wild flowering cherry <Emphasis Type="Italic">Prunus yedoensis</Emphasis> var. <Emphasis Type="Italic">nudiflora</Emphasis> (Rosaceae)

Prunus yedoensis Matsumura is one of the popular ornamental flowering cherry trees native to northeastern Asia, and its wild populations have only been found on Jeju Island, Korea. Previous studies suggested that wild P. yedoensis (P. yedoensis var. nudiflora) is a hybrid species; however, there is no solid evidence on its exact parental origin and genomic organization. In this study, we developed a total of 38 nuclear gene-based DNA markers that can be universally amplifiable in the Prunus species using 586 Prunus Conserved Orthologous Gene Set (Prunus COS). Using the Prunus COS markers, we investigated the genetic structure of wild P. yedoensis populations and evaluated the putative parental species of wild P. yedoensis. Population structure and phylogenetic analysis of 73 wild P. yedoensis accessions and 54 accessions of other Prunus species revealed that the wild P. yedoensis on Jeju Island is a natural homoploid hybrid. Sequence-level comparison of Prunus COS markers between species suggested that wild P. yedoensis might originate from a cross between maternal P. pendula f. ascendens and paternal P. jamasakura. Moreover, approximately 81% of the wild P. yedoensis accessions examined were likely F1 hybrids, whereas the remaining 19% were backcross hybrids resulting from additional asymmetric introgression of parental genotypes. These findings suggest that complex hybridization of the Prunus species on Jeju Island can produce a range of variable hybrid offspring. Overall, this study makes a significant contribution to address issues of the origin, nomenclature, and genetic relationship of ornamental P. yedoensis.