A hyperactive transposase of the maize transposable element activator (Ac)

Activator/Dissociation (Ac/Ds) transposable elements from maize are widely used as insertional mutagenesis and gene isolation tools in plants and more recently also in medaka and zebrafish. They are particularly valuable for plant species that are transformation-recalcitrant and have long generation cycles or large genomes with low gene densities. Ac/Ds transposition frequencies vary widely, however, and in some species they are too low for large-scale mutagenesis. We discovered a hyperactive Ac transposase derivative, AcTPase(4x), that catalyzes in the yeast Saccharomyces cerevisiae 100-fold more frequent Ds excisions than the wild-type transposase, whereas the reintegration frequency of excised Ds elements is unchanged (57%). Comparable to the wild-type transposase in plants, AcTPase(4x) catalyzes Ds insertion preferentially into coding regions and to genetically linked sites, but the mutant protein apparently has lost the weak bias of the wild-type protein for insertion sites with elevated guanine-cytosine content and nonrandom protein-DNA twist. AcTPase(4x) exhibits hyperactivity also in Arabidopsis thaliana where it effects a more than sixfold increase in Ds excision relative to wild-type AcTPase and thus may be useful to facilitate Ac/Ds-based insertion mutagenesis approaches.     

Probing electron transfer reactions between two azurins from Alcaligenes xylosoxidans GIFU 1051 with optically active Ru complexes as molecular recognition probes: Importance of the 43rd residue

Electron transfer reactions between optically-active Ru^II/III complexes incorporating (S)-/(R)-amino acids, and the two azurins, azurin-1 (az-1Cu) and azurin-2 (az-2Cu) isolated from Alcaligenes xylosoxidans GIFU 1051, have been studied to probe molecular recognition sites on the two azurins. The Ru^II/III complexes are K[Ru^II(L)(bpy)] and [Ru^III(L)(bpy)], and have a tripodal ligand (L) derived from the (S)-/(R)-amino acids, which are in turn exchanged for other functional substituent groups, such as (S)-/(R)-phenylalanine, -leucine, -valine, -alanine, and -glutamic acid (L = (S)-/(R)-BCMPA, -BCMLE, -BCMVA, -BCMAL, and -BCMGA). In the oxidation reaction of az-1Cu^I promoted by the Ru^III complexes, the kinetic parameters exhibited enantio- and stereo-selectivities, while the same reaction of az-2Cu^I was less enantio- and stereo-selective. These differences suggest that the processes of formation of the activated states are different for the two azurins. On the other hand, such a difference has not been observed for az-1 and az-2 with respect to the reduction reactions promoted by both azurins Cu^II by the Ru^II complexes within the experimental error. This suggests that the neutrality of the Ru complexes is important for precise molecular recognition of azurins. His117 has been proposed as the electron transfer site. The local structures in the vicinity of the His117 side chain in the two azurins, are essentially identical with the exception of the 43rd residue, Val43 and Ala43 for az-1 and az-2, respectively. Electron transfer reactions between Ru^III complexes and a mutant azurin, V43A-az-1, were also carried out. Interestingly, the activation parameters estimated were very similar to those of az-2, indicating that the 43rd residue acts as the electron transfer site in azurins and provides rationalization for the different mechanisms of az-1 and az-2 in redox reactions.     

Characterization of 10 MADS-box genes from Pyrus pyrifolia and their differential expression during fruit development and ripening

We cloned 10 Japanese pear (Pyrus pyrifolia) MIKC-type II MADS-box genes, and analyzed their expression during fruit development and ripening. PpMADS2-1 was APETALA (AP)1-like; PpMADS3-1 was FRUITFULL (FUL)/SQUAMOSA (SQUA)-like; PpMADS4-1 was AGAMOUS-like (AGL)6; PpMADS5-1 and PpMADS8-1 were SUPPRESSOR OF OVEREXPRESSION OF CONSTANS (SOC)-like; PpMADS9-1, PpMADS12-1, PpMADS14-1 and PpMADS16-1 were SEPALLATA (SEP)-like; while PpMADS15-1 was AGL/SHATTERPROOF (SHP)-like. Phylogenetic analysis showed their grouping into five major clades (and 10 sub-clades) that was consistent with their diverse functional types. Expression analysis in flower tissue revealed their distinct putative homeotic functional classes: A-class (PpMADS2-1, PpMADS3-1, PpMADS4-1, and PpMADS14-1), C-class (PpMADS15-1), E-class (PpMADS9-1, PpMADS12-1, and PpMADS16-1) and E (F)-class (PpMADS5-1 and PpMADS8-1). Differential gene expression was observed in different fruit tissues (skin, cortex and core) as well as in the cortex during the course of fruit development and ripening. Collectively, our results suggest their involvement in the diverse aspects of plant development including flower development and the course of fruit development and ripening.     

Identification of nucleopolyhedrovirus that infect Nymphalid butterflies Agraulis vanillae and Dione juno

Dione juno and Agraulis vanillae are very common butterflies in natural gardens in South America, and also bred worldwide. In addition, larvae of these butterflies are considered as pests in crops of Passiflora spp. For these reasons, it is important to identify and describe pathogens of these species, both for preservation purposes and for use in pest control. Baculoviridae is a family of insect viruses that predominantly infect species of Lepidoptera and are used as bioinsecticides. Larvae of D. juno and A. vanillae exhibiting symptoms of baculovirus infection were examined for the presence of baculoviruses by PCR and transmission electron microscopy. Degenerate primers were designed and used to amplify partial sequences from the baculovirus p74, cathepsin, and chitinase genes, along with previously designed primers for amplification of lef-8, lef-9, and polh. Sequence data from these six loci, along with ultrastructural observations on occlusion bodies isolated from the larvae, confirmed that the larvae were infected with nucleopolyhedroviruses from genus Alphabaculovirus. The NPVs from the two different larval hosts appear to be variants of the same, previously undescribed baculovirus species. Phylogenetic analysis of the sequence data placed these NPVs in Alphabaculovirus group I/clade 1b.     

Population Dynamics of Plant Nematodes in Cultivated Soil: Effect of Summer Cover Crops in Old Agricultural Land

In a 6-year cover crop sequence study, nematode population densities varied with different cover crops. Millet favored rapid increase of Belonolaimus longicaudatus and supported relatively large numbers of Pratylenchus brachyurus. Beggarweed and ''Coastal'' bermudagrass favored rapid increase of B. Iongicaudatus and to a lesser extent P. brachyurus and Trichodorus christiei. Hairy indigo and Crotalaria supported relatively large numbers of P. brachyurus but suppressed B. longicaudatus. Hairy indigo also supported increases of T. christiei and Xiphinema americanum. Marigold did not favor development of any parasitic nematode species present. Tomato transplant yield was inversely related to nematode population, particularly to B. Iongicaudatus. Largest yields were obtained from plots with smallest numbers of B. longicaudatus and smallest yields were from plots with largest numbers of B. longicaudatus.     

Steinernema feltiae: ammonia triggers the emergence of their infective juveniles

Entomopathogenic nematodes complete their life cycles inside dead insects. The emergence of new infective juveniles from the cadaver has been attributed (but never demonstrated) to food depletion or to the accumulation of metabolites from the breakdown of the host's tissues. Here we give evidence that emergence is triggered by ammonia, a product of nematode defecation. We found that the emergence of Steinernemafeltiae infective juveniles from Galleriamellonella cadavers was stimulated by a particular level of ammonia. Emergence was delayed when ammonia in the cadaver was decreased and was prompted when increased. These findings will further improve the understanding of the nematode life cycle. Here we speculate that production of infective juveniles can be mediated by ammonia and work in a manner analogous to that of the dauer recovery inhibiting factor (DRIF) in Caenorhabditiselegans.     

Population Dynamics of Plant Nematodes in Cultivated Soil: Effect of Summer Cover Crops in Newly Cleared Land

Five nematode species were studied for ability to develop on seven summer cover crops in rotation with tomato transplants grown every third year. Increase of Tylenchorhynchus claytoni, Trichodorus christiei, Pratylenchus brachyurus, Helicotylenchus dihystera, and Xiphinema americanum in newly cleared soil varied with different cover crops. No substantial nematode population increases occurred until the third summer of crop growth. All species except X. americanum and H. dihystera developed best on sudangrass and millet. Crotalaria caused substantial increase of H. dihystera and P. brachyurus but suppressed the other species. Marigold suppressed all species except X. americanum which increased substantially on marigold during the 5th year. Cotton favored rapid increase of T. christiei, and moderate increases of all species except T. claytoni which was suppressed. Beggarweed favored moderate increases of T. christiei and H. dihystera but suppressed the other species. Hairy indigo favored rapid increase of H. dihystera, moderate increases of T. christiei and X. americanum, and suppressed the other species. Number of marketable transplants was reduced after 2 years of sudangrass and cotton; these crops favored increases of T. christiei and T. claytoni. The better cover crops prevented increases of most plant parasitic nematodes in land cropped to tomato, a suitable host.     

Leishmania (Viannia) panamensis: An in vitro assay using the expression of GFP for screening of antileishmanial drug

Promastigotes of Leishmania (Viannia) panamensis were successfully transfected with p6.5-egfp to express green fluorescent protein. The transfectants remained infective to macrophages, providing an in vitro model for screening antileishmanial drugs. This was demonstrated by flow cytometry of macrophage-associated GFP after exposure of infected cultures to known antileishmanial drugs, i.e. amphotericin B and glucantime^®. Fluorescence of GFP diminished progressively from infected cells with increasing drug concentrations used in both cases. The availability of this fluorescent assay for infection of macrophages by L. (V.) panamensis facilitates drug discovery program for the Viannia species, which differ significantly from those of the Leishmania subgenus.     

生境不完全重叠的两种鲤科鱼类耐低氧及运动能力比较

以乌江流域亲缘关系近,但分布并不完全重叠的马口鱼(Opsariichthys bidens)和宽鳍鱲(Zacco platypus)作为实验对象,分别考察这两种实验鱼的低氧耐受及游泳运动能力。将野外采回的实验鱼置于(25±1)℃条件下,分别测定两种鱼的临界氧分压(Pcrit)、水面呼吸(ASR)、失去平衡点(LOE)以及在不同溶氧水平(8.0、4.0和2.0 mg/L)下的临界游泳速度(Ucrit)和活跃耗氧率(MO2active)。研究发现:马口鱼的Pcrit(2.44±0.20)mg/L显著高于宽鳍鱲(1.86±0.10)mg/L(P=0.031)。但马口鱼的50%ASR(1.23±0.16)mg/L显著低于宽鳍鱲(1.97±0.11)mg/L(P=0.023);马口鱼的50%LOE(0.84±0.01)mg/L同样显著低于宽鳍鱲(0.97±0.02)mg/L(P=0.004)。宽鳍鱲在8.0和4.0 mg/L下的游泳能力显著高于马口鱼,然而马口鱼和宽鳍鱲的Ucrit均随测定溶氧水平的下降而显著降低(P0.01);宽鳍鱲和马口鱼的运动耗氧率均随水流速度的增加而呈现指数增加,但随测定溶氧水平的降低运动耗氧曲线变得相对平缓,尤其是在溶氧为4.0 mg/L时马口鱼的运动耗氧曲线与宽鳍鱲相比越发平缓;两种实验鱼的MO2active随溶氧水平下降的变化趋势与Ucrit相似(P0.001)。结果表明:两种野外生存的实验鱼不仅在低氧耐受能力方面存在显著差异,而且两者的游泳运动能力也有不同表现,这很有可能与其遗传特征、生存环境及生态习性相关。     

Association of glutathione S-transferase gene polymorphisms (GSTM1 and GSTT1) of vitiligo in Korean population

Vitiligo is an acquired pigmentary disorder of the skin involving melanocyte dysfunction. It has been reported that melanocyte impairment could be related to increased oxidative stress. The glutathione S-transferases (GSTs) are group of polymorphic enzymes that are important in protection against oxidative stress. To find the relationship between GSTM1 and GSTT1 polymorphisms with vitiligo susceptibility, GSTM1 and GSTT1 (homozygous deletion vs. non-deleted) polymorphisms between vitiligo patients (n=310) and healthy controls (n=549) were analyzed. We observed significant association in null alleles of the GSTM1 (P<0.001, OR=2.048, 95% CI=1.529-2.743). GSTM1 null type was also statistically different between two vitiligo subtypes and controls (Focal P<0.001, OR=2.224, 95% CI=1.499-3.298; Generalized P=0.001, OR=1.974, 95% CI=1.342-2.904). However, no significant association in GSTT1 (P=0.869, OR=1.024, 95% CI=0.775-1.353) was observed with vitiligo. In combined analysis of GSTM1 and GSTT1, both null type and GSTM1/GSTT1 (null/present) group showed significant differences between controls and vitiligo patients. These results suggest that GSTM1 null type might be associated with vitiligo susceptibility in Korean population.     

A cis-encoded sRNA controls the expression of fabH2 in Yersinia

YsrH is a novel cis-encoded sRNA located on the opposite strand to fabH2, which is essential for fatty acid biosynthesis in bacteria. In this study, YsrH-mediated regulation of fabH2 expression was investigated in Yersinia pseudotuberculosis. Constitutive and inducible over-expression of YsrH decreased the mRNA level of fabH2, while expression of downstream fabD and fabG remained unaffected. Polynucleotide phosphorylase (PNPase) also played an important role in this regulation process by mediating YsrH decay in the exponential phase. Thus, our data defines a cis-encoded sRNA that regulates fatty acid synthesis via a regulatory mechanism also involving PNPase.     

Relationships of plant pathogenic enterobacteria based on partial atpD, carA, and recA as individual and concatenated nucleotide and peptide sequences

Relationships of the genera in the Enterobacteriaceae containing plant pathogenic species: Brenneria, Dickeya, Enterobacter, Erwinia, Pantoea, Pectobacterium, and Samsonia, were investigated by comparison of their nucleotide and peptide sequences of atpD, carA, recA, and the concatenated sequences. Erwinia spp. and Pantoea spp., with Pectobacterium cypripedii, formed a group distinct from other pathogenic taxa. Pectobacterium, Brenneria, Dickeya, and Samsonia formed a contiguous clade. Samsonia was usually concurrent with Pectobacterium. Most Brenneria were also close to Pectobacterium, suggesting that these three taxa might be better represented as a single genus. Brenneria quercina was not closely associated with other members of this genus and may represent a separate genus. The sequences representing Dickeya were distinct, further supporting the generic status of the taxon. Plant pathogenic Enterobacter spp. display such sequence variability that few definite conclusions as to their specific placement could be made. These data highlight the difficulty of drawing reliable and robust taxonomic conclusions based on comparative analysis of sequence data without some independent criterion to calibrate a scale for diversity.     

Production of (2S,3S)-2,3-butanediol and (3S)-acetoin from glucose using resting cells of Klebsiella pneumonia and Bacillus subtilis

Production of highly pure (2S,3S)-2,3-butanediol ((2S,3S)-2,3-BD) and (3S)-acetoin ((3S)-AC) in high concentrations is desirable but difficult to achieve. In the present study, glucose was first transformed to a mixture of (2S,3S)-2,3-BD and meso-2,3-BD by resting cells of Klebsiella pneumoniae CICC 10011, followed by biocatalytic resolution of the mixture by resting cells of Bacillus subtilis 168. meso-2,3-BD was transformed to (3S)-AC, leaving (2S,3S)-2,3-BD in the reaction medium. Using this approach, 12.5 g l(-1) (2S,3S)-2,3-BD and 56.7 g l(-1) (3S)-AC were produced. Stereoisomeric purity of (2S,3S)-2,3-BD and enantiomeric excess of (3S)-AC was 96.9 and 96.2%, respectively.     

Stereoselectivity of the demethylation of nicotine piperidine homologues by Nicotiana plumbaginifolia cell suspension cultures

The metabolism of (R,S)-N-methylanabasine and (R,S)-N-methylanatabine has been studied in a cell suspension culture of Nicotiana plumbaginifolia. Both substrates are effectively demethylated, anabasine or anatabine, respectively, accumulating in the medium. Similarly, there is strong stereoselectivity for the (R)-isomers of both substrates. The kinetics of metabolism of (R,S)-N-methylanabasine differ significantly from those of nicotine in that no further degradation of the initial demethylation product occurs. (R,S)-N-Methylanatabine, however, shows kinetics closer to those of nicotine, with loss of alkaloid from the system. Further more, (R,S)-N-methylanabasine does not diminish (S)-nicotine demethylation, indicating a lack of competition. However, the metabolism of (S)-nicotine is affected by the presence of (R,S)-N-methylanabasine. Hence, the demethylation of the piperidine homologues of nicotine is seen to be similar but not identical to that of the pyridine analogues. The implications of these different metabolic profiles in relation to the demethylation activity are discussed.     

New inhibitors of colony spreading in Bacillus subtilis and Bacillus anthracis

We have recently characterized sliding motility in Bacillus subtilis strains that lack functional flagella, and here describe the discovery of inhibitors of colony spreading in these strains as well as the aflagellate pathogen, Bacillus anthracis. Aflagellate B. subtilis strains were used to screen for new types of antibacterials that might inhibit colony spreading on semi-solid media. From a diverse set of organic structures, p-nitrophenylglycerol (NPG), an agent used primarily in clinical laboratories to control Proteus swarming, was found to inhibit colony spreading. The four stereoisomers of NPG were synthesized and tested, and only the 1R,2S-(1R-anti) and 1R,2R-(1R-syn) NPG isomers had significant activity in a quantitative colony-spreading assay. Twenty-six NPG analogs and related structures were synthesized and tested to identify more active inhibitors. p-Methylsulfonylphenylglycerol (p-SPG), but not its ortho or meta analogs, was found to be the most effective of these compounds, and synthesis and testing of all four p-SPG stereoisomers showed that the 1R-anti-isomer was the most active with an average IC(50) of 16 μM (3-5 μg mL(-1)). For B. anthracis, the colony-spreading IC(50) values for 1R-anti-SPG and 1R-anti-NPG are 12 μM (2-4 μg mL(-1)) and >150 μM, respectively. For both Bacillus species tested, 1R-anti-SPG inhibits colony spreading of surface cultures on agar plates, but is not bacteriostatic or bacteriocidal in liquid cultures. Work is in progress to find the cellular target(s) of the NPG/SPG class of compounds, since this could lead to an understanding of the mechanism(s) of colony spreading as well as design and development of more potent inhibitors for the control of B. anthracis surface cultures.     

黑龙江省东完达山地区东北虎猎物种群现状及动态趋势

猎物种群丰度是限制虎分布和数量的关键因子,因此猎物种群密度监测和估算是虎保护的重要内容之一。应用采用大样方法,地理信息系统技术和多元统计分析,研究了黑龙江东完达山东部地区东北虎猎物种群(马鹿、狍子和野猪)现状及动态变化趋势。结果表明:研究地区马鹿的种群平均密度为(0.2010±0.0270)只/km²、狍子的平均种群密度为(0.4980±0.0436)只/km²、野猪的平均种群密度为(0.3423±0.0275)只/km²。单因素方差分析表明,在相同生境下,3种有蹄类密度在在阔叶混交林中和杂木林中差异极为显著;不同的生境,3种猎物的猎物的密度也存在着显著差异。相关分析表明马鹿密度和野猪密度程正相关,而马鹿密度和狍子密度、狍子密度和野猪密度则不相关。 同1989年该地区东北虎猎物种群相比:1989-2002年的13 a时间内马鹿的年平均递减率为13.48%、狍子的年平均递减率为12.69%、野猪的年平均递减率为1.89%。     

Lavandula angustifolia essential oil as a novel and promising natural candidate for tick (Rhipicephalus (Boophilus) annulatus) control

Lavandula angustifolia is a well known herbal medicine with a variety of useful properties, including its acaricidal effect. This experiment was carried out to study the bioacaricidal activity of L. angustifolia essential oil (EO) against engorged Rhipicephalus (Boophilus) annulatus (Acari; Ixodidae) females. For this purpose six serial concentrations (0.5, 1.0, 2.0, 4.0, 6.0 and 8.0% w/v) of L. angustifolia EO were used. There was considerable mortality in concentrations more than 4.0% although there were no differences between 6.0 and 8.0% in all measured criteria. The mortality rate 24 h after inoculation was 73.26 and 100% in groups treated with 4.0 and 8.0% EO, respectively. Lavender EO also reduced tick egg weight in a concentration-dependent manner. The amount of eggs produced varied from 0.12 g (at 0.5% EO) to 0.00 g (at 8.0% EO) but did not differ statistically from the control. L. angustifolia EO caused 100% failure in egg laying at 6.0 and 8.0% whereas this value in the control group was zero. A positive correlation between L. angustifolia EO concentration and tick control, assessed by relative mortality rate and egg-laying weight, was observed by the EO LC/EC₅₀, which, when calculated using the Probit test, was 2.76-fold higher than the control. Lavender is a promising acaricidal against R. (B.) annulatusin vitro.     

At least two regions of the oncoprotein Tre2 are involved in its lack of GAP activity

The product of the human Tre2 oncogene is structurally related to the Ypt/Rab GTPase-activating proteins (Ypt/Rab GAPs). Particularly, the oncoprotein shares with the yeast proteins Msb3p and Msb4p, and with the human protein RN-tre the highly conserved TBC domain, forming the catalytically active domain of Ypt/Rab GAPs. Yet, the Tre2 oncogene seems to encode a nonfunctional Rab GAP. As regions flanking the TBC domain may be crucial for catalytic activity, regions located N- and C-terminally with respect to this domain were explored. For this, chimeric proteins created by sequence exchanges between the Tre2 oncoprotein and RN-tre were tested for their ability to replace functionally the Msb3p and Msb4p proteins in double-mutant yeast cells. These complementation experiments revealed, in addition to the TBC domain, a second Tre2 region involved in the oncoprotein's lack of GAP activity: a 93-aa region flanking the TBC domain on the C-terminal side.     

Allele-specific effects of PDF2 on floral morphology in Arabidopsis thaliana

The class IV Homeodomain-leucine zipper (HD-ZIP IV) gene family includes several genes that are functionally significant in epidermal development. Our recent study revealed that double mutants of the epidermis-expressed HD-ZIP IV members, PROTODERMAL FACTOR2 (PDF2) in combination with some HOMEODOMAIN GLABROUS (HDG, pronounced “hedge”) genes, affect stamen development and specification of petal and stamen identity, possibly in a non cell-autonomous manner. However, the effect of the pdf2 mutations on the floral development was largely different depending on T-DNA insertion locations: pdf2–1 hdg flowers exhibited homeotic conversion of petals and stamens, while pdf2–2 hdg flowers had only a reduced number of stamens. Here, we used 2 additional pdf2 alleles to make double mutants and found that their floral phenotypes were rather similar to those of pdf2–2 hdg. The allele-specific effect caused by pdf2–1, which carries a T-DNA in a steroidogenic acute regulatory protein-related lipid transfer (START) domain-encoding region, suggests the importance of the START domain in proper function of HD-ZIP IV proteins.