Size structures and comparative phenology of syntopic populations of <Emphasis Type="Italic">Hirudo verbana</Emphasis> and <Emphasis Type="Italic">Hirudo medicinalis</Emphasis> in eastern Ukraine

Ranges of different species of medicinal leeches (Hirudo spp.) are nearly mutually exclusive with very few areas of overlapping. This biogeographical pattern suggests different ecological requirements for the two species. However, two habitats in Hungary and Ukraine where populations of H. verbana and H. medicinalis coexist were found. The aim of this research was to reveal differences in size structures and phenologies between the two species in a steppe pond with two populations. The study was conducted from March to September in 2009 and 2010 in Horila Dolyna, Kharkiv Region. Leeches were collected several times during the season, weighed and released. The abundance of H. verbana was always higher. The Mann-Whitney U-test and the Wilcoxon test were applied. Significant differences between sequential samples within the species were found. The large weight classes of H. verbana emerged by the end of summer and caused a higher variance in contrast to H. medicinalis. This suggests the higher growth rate and the more r-strategic biology of H. verbana in comparison with the largely K-strategic H. medicinalis.     

Cannibalism in a Population of the Medicinal Leech (<Emphasis Type="Italic">Hirudo medicinalis</Emphasis> L.)

Medicinal leeches (Hirudo medicinalis L.) were maintained in large ponds in a commercial leech farm at Biebertal, Germany. The feeding of hungry adult leeches was performed on representative individuals that were placed on cloth soaked with mammalian blood obtained from a local butchery (pig, Sus scrofa). In a second set of experiments, cane toads (Bufo marinus) were used as host organisms. The leeches rapidly attached to the toads, explored the body and sucked blood. After feeding, the fully engorged leeches were placed into the pond or an aquarium. In this artificial habitat, the satiated leeches were attacked by hungry conspecifics, sucked off, and killed. This observation demonstrates that H. medicinalis must be classified as a cannibalistic annelid.     

Neuro-and psychotropic activity of <Emphasis Type="Italic">N</Emphasis>-uronoylamino acids and <Emphasis Type="Italic">N</Emphasis>-uronoylpeptides

Peculiarities of the rat behavior were studied in a series of experimental stress models after a systemic administration of new N-uronoyl derivatives of amino acids. The psychotropic effect was shown to be determined by the nature of the amino acid fragment. N-(1,2:3,4-Di-O-isopropylidene-α-D-galactopyraneuronoyl)-glycylglycine exhibited an anxiolytic effect more pronounced than that of pyracetam, whereas N-(1,2:3,4-di-O-isopropilidene-α-D-galactopyranuronoyl)-glycylglutamic acid has antidepressant action stronger than that of amitriptyline. Mechanisms for the psychotropic effects of the examined derivatives are discussed.     

A new <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> deletion mutant <Emphasis Type="Italic">apetala1-20</Emphasis>

A new deletion allele of the APETALA1 (AP1) gene encoding a type II MADS-box protein with the key role in the initiation of flowering and development of perianth organs has been identified in A. thaliana. The deletion of seven amino acids in the conserved region of the K domain in the ap1-20 mutant considerably delayed flowering and led to a less pronounced abnormality in the corolla development compared to the weak ap1-3 and intermediate ap1-6 alleles. At the same time, a considerable stamen reduction has been revealed in ap1-20 as distinct from ap1-3 and ap1-6 alleles. These data indicate that the K domain of AP1 can be crucial for the initiation of flowering and expression regulation of B-class genes controlling stamen development.     

Identification of LMW Glutenin-Like Genes from <Emphasis Type="Italic">Secale sylvestre</Emphasis> Host

Three low-molecular-weight (LMW) glutenin-like genes (designated as Ssy1, Ssy2, and Ssy3) from Secale sylvestre Host were isolated and characterized. The three genes consist of a predicted highly conservative signal peptide with 20 amino acids, a short N-terminal region with 13 amino acids, a highly variable repetitive domain and a less variable C-terminal domain. The deduced amino acid sequences of the three genes were the LMW-m type due to a methionine residue at the N-terminus. The phylogenetic analysis indicated that the prolamin genes could be perfectly clustered into five groups, including HMW-GS, LMW-GS, α/β-, γ-, and κ-prolamin. The LMW glutenin-like genes of S. sylvestre were more orthologous with the LMW-GS genes of wheat and B hordein genes of barley, which also had been confirmed by the homology analysis with the LMW-GS of wheat at Glu-A3, Glu-B3, and Glu-D3 loci. These results indicated that a chromosome locus (designated as Glu-R3) might be located on the R genome of S. sylvestre with the functions similar to the Glu-3 locus in wheat and its related species.     

The earliest samples of <Emphasis Type="Italic">Hymenoscyphus albidus</Emphasis> vs. <Emphasis Type="Italic">H. fraxineus</Emphasis> in Estonian mycological herbaria

In 2006, the alien and pathogenic Hymenoscyphus fraxineus was differentiated by molecular methods from the long recognized saprotrophic H. albidus . Today, H. fraxineus seems to have replaced H. albidus in several countries, but the exact year of arrival of H. fraxineus in northern Europe is still debated. Investigations of herbaria specimens might help to ascertain it. Before the epidemic, H. albidus was not of significant interest to mycologists in the Baltic area and was not eagerly sampled and deposited. Nevertheless, the TAAM herbarium in Estonia holds 13 putative H. albidus specimens that were collected 1966–2006. Using newly developed species-specific PCR primers to differentiate between H. albidus and H. fraxineus, all available herbarium samples from TAAM were identified. In addition, the primers were also tested on pure cultures and ascocarps of H. fraxineus and H. albidus, symptomatic petioles of F. excelsior, pure cultures of 10 non-target fungal species, and 10 different soil samples. With the exception of the oldest specimen in TAAM from 1966 (collected in Lithuania), all herbarium samples were identified as H. fraxineus. The first record of H. fraxineus in Estonia dates back to 1997 and apparently represents the oldest record of the species in Europe. In spite of this, symptoms based on reliable observations were not detected until 2003 in Estonia. This temporal shift is difficult to explain. Possible reasons may represent (i) overlooking of symptoms during the first years; (ii) typical lag phase at the start of an epidemic; (iii) an abrupt shift towards higher virulence of H. fraxineus; or (iv) other environmental (climatic) factors. Closer investigations are needed to disentangle these possible reasons.     

Regulating the regulators: responses of four plant growth regulators during clonal propagation of <Emphasis Type="Italic">Lachenalia montana</Emphasis>

Geraniol, the precursor of terpenoid indole alkaloids can be converted to the 10-hydroxy geraniol by the function of geraniol 10-hydroxylase. In our study, for the first time, a full-length cytochrome P450 monooxygenase (P450) geraniol 10-hydroxylase (PgCYP76C9) cDNA was isolated and characterized from Panax ginseng Meyer. The gene has an open reading frame (ORF) of 1503 base pairs and encodes a precursor protein of 501 amino acids residues. The calculated molecular mass of the protein is approximately 56.3 kDa with a predicated isoelectric point of 8.45. Amino acid identities between PgG10H and other P450s of the CYP76 family in the database had revealed that the deduced amino acid of PgG10H sharing a higher sequence homology with geraniol 10-hydroxylase-like proteins encoded by Cinchona calisaya and Lonicera japonica. We implemented a molecular modeling method to evaluate the possible interaction of geraniol with PgG10H active site. Our finding showed that the geraniol was the potential ligand for PgG10H in P. ginseng. Expression of PgG10H gene was tissue-regulated and showed high expression in 3-year-old ginseng flowers and roots. Expression of PgG10H was differentially induced in ginseng, not only during Pseudomonas syringae infection and wounding but also after exposure to methyl jasmonate and salt stress. Furthermore, overexpression of the newly identified ginseng geraniol 10-hydroxylase P450 gene in Arabidopsis caused terpenoid indole alkaloid dihydrositsirikine production and also conferred enhanced resistance to P. syringae.     

Isolation and characterization of lectin from the surface of <Emphasis Type="Italic">Grifola frondosa</Emphasis> (Fr.) S.F.Gray mycelium

From the surface of the dikaryotic mycelium of the xylotrophic basidiomycete Grifola frondosa 0917 a lectin has been isolated with a molecular mass of 68 ± 1 kDa, consisting of two subunits of 33–34 kDa each. The lectin is a hydrophilic glycoprotein with the protein: glycan ratio of 3: 1. It exhibits high affinity to native rabbit erythrocytes and to human erythrocytes of the 0 blood group, but not to trypsin-treated ones. The hemagglutination (HA) caused by lectin was not blocked by any of the 25 tested mono-, di-, and amino sugars; it was also not blocked by some of glyco derivatives. Only 13.9 μg/ml of the homogeneous preparation of a polysaccharide, a linear D-rhamnan with the structure of the repeated component →2)-β-D-Rhap-(1→3)-α-D-Rhap-(1→3)-α-D-Rhap-(1→2)-α-D-Rhap-(1→2)-α-sD-Rhap-1(→ blocked hemagglutination completely. The analysis of the amino acid composition of the lectin showed the greatest percentage of amino acids with positively charged R groups, arginine, lysine, and histidine, as well as the complete absence of sulfurcontaining amino acids, cysteine, and methionine. D-glucose and D-glucosamine were detected in the carbohydrate part. Original Russian Text ? L.V. Stepanova, V.E. Nikitina, A.S. Boiko, 2007, published in Mikrobiologiya, 2007, Vol. 76, No. 4, pp. 488–493.     

Steroid Compounds from Far Eastern Starfishes <Emphasis Type="Italic">Henricia aspera</Emphasis> and <Emphasis Type="Italic">H. tumida</Emphasis>

Six new natural compounds were isolated from two Far Eastern starfish species, Henricia aspera and H. tumida, collected in the Sea of Okhotsk. Two new glycosylated steroid polyols were obtained from H. aspera: asperoside A and asperoside B, which were shown to be (20R,24R, 25S)-3-O-(2,3-di-O-methyl-β -D-xylopyranosyl)-24-methyl-5α-cholest-4-ene-3β, 6β,8,15α,16β,26-hexaol and (20R, 24R,25S,22E)-3-O-(2,4-di-O-methyl-β-D-xylopyranosyl)-24-methyl-5α-cholest-22-ene-3β,4β,6β,8,15α,26-hexaol, respectively. Two other glycosylated polyols, tumidoside A, with the structure elucidated as (20R, 22E)-3-O-(2,4-di-O-methyl-β -D-xylopyranosyl)-26,27-dinor-24-methyl-5α-cholest-22-ene-3β,4β,6β,8,15α,25-hexaol, and tumidoside B, whose structure was elucidated as (20R,24S)-3-O-(2,3-di-O-methyl-β-D-xylopyranosyl)-5α-cholestan-3β,4β,6β,8,15α,24-hexaol, were isolated from the two starfish species. (20R, 24S)-5α-Cholestan-3β,6β,15α,24-tetraol and (20R, 24S)-5α-cholestan-3β,6β,8,15α,24-pentaol were identified only in H. tumida. The known monoglycosides henricioside H1 and laeviuscolosides H and G were also identified in both species.     

Deletion of <Emphasis Type="Italic">ldh</Emphasis>A and <Emphasis Type="Italic">ald</Emphasis>H genes in <Emphasis Type="Italic">Klebsiella</Emphasis><Emphasis Type="Italic">pneumoniae</Emphasis> to enhance 1,3-propanediol production

Objectives

To improve 1,3-propanediol (1,3-PD) production and reduce byproduct concentration during the fermentation of Klebsiella pneumonia.

Results

Klebsiella. pneumonia 2-1ΔldhA, K. pneumonia 2-1ΔaldH and K. pneumonia 2-1ΔldhAΔaldH mutant strains were obtained through deletion of the ldhA gene encoding lactate dehydrogenase required for lactate synthesis and the aldH gene encoding acetaldehyde dehydrogenase involved in the synthesis of ethanol. After fed-batch fermentation, the production of 1,3-PD from glycerol was enhanced and the concentrations of byproducts were reduced compared with the original strain K. pneumonia 2-1. The maximum yields of 1,3-PD were 85.7, 82.5 and 87.5 g/l in the respective mutant strains.

Conclusion

Deletion of either aldH or ldhA promoted 1,3-PD production in K. pneumonia.

     

Molecular cloning and characterization of a PR-5 like protein gene from <Emphasis Type="Italic">Brassica campestris</Emphasis> ssp. <Emphasis Type="Italic">chinensis</Emphasis>

Downy mildew caused by Hyaloperonospora parasitica is a serious fungal disease in non-heading Chinese cabbage (Brassica campestris L. ssp. chinensis Makino). Pathogenesis-related 5 (PR-5) genes play an important role in plant resistance to disease invasion. In this study, a gene encoding pathogenesis-related 5-like (PR-5L) protein, named BcPR-5L, was successfully cloned from non-heading Chinese cabbage. The cDNA sequence of BcPR-5L was 747 bp in length. It encoded a protein of molecular mass of 25.78 kDa, an isoelectric point of 4.42, and containing 248 amino acids. Multiple sequence alignment indicated that BcPR-5L protein was highly homologous to other PR-5L proteins identified in 13 different species, with the highest homology to Brassica rapa. We analyzed the subcellular localization of BcPR-5L protein by using onion epidermal cells and found that it was localized in the membrane. Real time quantitative PCR analyses revealed that the expression of BcPR-5L gene was significantly upregulated after H. parasitica infection, and the expression in the resistant cultivar was higher than that in the susceptible cultivar. In summary, our data suggest that BcPR-5L gene may play an important role in the resistance of non-heading Chinese cabbage to H. parasitica infection.     

Heterologous expression of a novel <Emphasis Type="Italic">Poa pratensis</Emphasis> gibberellin 2-oxidase gene, <Emphasis Type="Italic">PpGA2ox</Emphasis>, caused dwarfism,late flowering,and increased chlorophyll accumulation in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Gibberellin 2-oxidases (GA2oxs) irreversibly convert bioactive gibberellins (GAs) and their immediate precursors into inactive GAs via 2-β hydroxylation and so regulate gibberellin content in plants. However, to the best of our knowledge, little has been known about the GA2oxs and its function in cool season turfgrass Poa pratensis. In this study, rapid amplification of cDNA end (RACE) was employed to isolate PpGA2ox from P. pratensis. The open reading frame of PpGA2ox was 1 047 bp in length, corresponding to 348 amino acids. PpGA2ox was localized in both nucleus and cytoplasm. The expression of PpGA2ox could be up-regulated by 10 μM gibberellic acid, 5 μM methyl jasmonate, or 10 μM indole-3-acetic acid. In addition, its native promoter could drive GUS expression in both leaf apex and shoot apical region. Moreover, overexpression of PpGA2ox in Arabidopsis led to GA-deficiency leading to dwarf phenotype, delayed flowering time, and increased chlorophyll content. Our study suggests that PpGA2ox could be a candidate gene for breeding new cultivars of P. pratensis.     

Overexpression of <Emphasis Type="Italic">SsDGAT2</Emphasis> from <Emphasis Type="Italic">Sapium sebiferum</Emphasis> (L.) Roxb Increases Seed Oleic Acid Level in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Sapium sebiferum (L.) Roxb is one of the most important oil trees in China. Diacylglycerol acyltransferases (DGATs) esterify sn-1, 2-diacylglycerol with a long-chain fatty acyl-CoA, the last step and the rate-limiting step of triacylglycerol (TAG) biosynthesis in prokaryotic and eukaryotic organisms. At least 74 DGAT2 sequences from 61 organisms have been identified, but the SsDGAT2 gene had not been reported to date. To clarify the function of SsDGAT2, we cloned the CDS (rapid amplification of cDNA end) of SsDGAT2 by RACE technology. The full-length CDS of SsDGAT2 contains 1011 bp and encodes a protein of 336 amino acids. Recombinant SsDGAT2 restored TAG biosynthesis to the yeast strain Saccharomyces cerevisiae H1246 TAG-deficient mutant and preferentially incorporated unsaturated C18 fatty acids into lipids. To investigate the biotechnological potential of SsDGAT2, it was expressed under the control of the 35S promoter in Arabidopsis Col-4. The oleic acid content increased by 50 % in transgenic plants relative to the control. The results indicated that most of the oleic acid increase was at the expense of linolenic acid (18:3) content, which suggests that high-oleic-acid-content seeds can be created by the overexpression of SsDGAT2 in S. sebiferum (L.) Roxb.     

Molecular characterization of <Emphasis Type="Italic">bmyC</Emphasis> gene of the mosquito pupicidal bacteria, <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> (VCRC B483) and in silico analysis of bacillomycin D synthetase C protein

A strain of Bacillus amyloliquefaciens (VCRC B483) exhibiting mosquito pupicidal, keratinase and antimicrobial activities was isolated from mangrove forest ecosystem of Andaman and Nicobar Islands. Molecular characterization of the strain showed the presence of lipopeptide encoding bmyC gene. Phylogenetic tree based on protein sequence of this gene exhibited homology with mycosubtilin synthetase of Bacilus atropheus and Iturin synthetase of Bacillus subtilis and B. amyloliquefaciens. This is the first report on the evolutionary conservation of amino acids concerned with the function and structure of bmyC protein of B. amyloliquefaciens. The presence of valine at the 1197th position in our strain was found to be unique and different from the existing strains of B. subtilis and B. amyloliquefaciens. Molecular modelling studies revealed significant changes in the structure of epimerization domain of the bmyC protein with A1197V variation. Crude metabolite of this strain exhibited antifungal activity against Fusarium sp. and Carvularia sp.     

Taste attractiveness of free amino acids and their physicochemical and biological properties (as exemplified by fishes)

Using fishes (32 species, 11 families) as an example, the relationship between the taste attractiveness of free amino acids (L-isomers) and their physicochemical and biological properties was analyzed. It was shown that essential amino acids, most nutritionally required for an organism, have lower taste attractiveness for fishes than nonessential amino acids. Only in 6 of the 32 tested species (sunbleak Leucaspius delineatus, European minnow Phoxinus phoxinus, dace Leuciscus leuciscus, chub Leuciscus cephalus, blue gourami Trichopodus trichopterus, pearl gourami Trichopodus leerii) the relationship between the taste attractiveness and molecular weight of amino acids was supported statistically, being negative in all cases. Only in 2 species, a statistically significant correlation between the taste properties of amino acids and the dissociation constant (K₁) was found, positive in the stone loach Barbatula barbatula and negative in the lake char Salvelinus namaycush. A positive correlation between taste preferences and the magnitude of the isoelectric point (pI) of amino acids was found in one species (roach Rutilus rutilus) and a negative correlation in 2 species (brown trout Salmo trutta and Arctic char Salvelinus alpinus erythrinus). A statistically significant correlation between the taste attractiveness and water solubility of amino acids was revealed in 2 species (chum salmon Oncorhynchus keta and navaga Eleginus nawaga), negative in both cases. The flavor, which stimulates food intake, was found to be more often intrinsic to acidic and polar uncharged than basic and nonpolar amino acids, L- than D-isomers, amino acids with an amino group at the α- than β-position. Amino acids are more attractive than their salts. Aromatic amino acids are much less attractive than S-containing or acyclic amino acids. Thus, in most fish species there is no or weak relationship between the taste attractiveness of free amino acids and many of their physical, chemical and biological properties, suggesting a mediated character of this relationship and/or its poor detectability.     

Chemical defences of native European coccinellid eggs against intraguild predation by the invasive Asian coccinellid, <Emphasis Type="Italic">Harmonia axyridis</Emphasis> (Pallas)

Harmonia axyridis (Pallas) is a coccinellid of Asian origin that has recently invaded substantial parts of Europe and is suspected to affect native coccinellid populations through intraguild predation and competition for food. Previous work has shown that two species from the Calvia genus appeared to be well protected against H. axyridis predation. To deepen our understanding on chemical protection of Calvia spp. and the predation risk by H. axyridis, we tested for susceptibility and palatability of Calvia spp. and H. axyridis eggs against predation by H. axyridis neonate larvae. Results show that eggs of C. quatuordecimguttata were mostly not eaten by H. axyridis, while eggs of the congeneric C. decemguttata were found to be largely unprotected against predation by the invasive coccinellid. We also observed that H. axyridis first instars successfully cannibalized on conspecific eggs. Removing the surface chemicals from C. quatuordecimguttata eggs resulted in significantly reduced protection from being preyed upon by H. axyridis, while applying these extracts onto C. decemguttata and H. axyridis eggs resulted in increased protection against H. axyridis larvae. The importance of surface chemicals in the interactions between H. axyridis and native coccinellids was confirmed by GC–MS analysis, showing a high diversity of hydrocarbons located on the surface of C. quatuordecimguttata eggs, i.e. more than twice as many when compared to C. decemguttata. Survival of H. axyridis larvae feeding on eggs of C. quatuordecimguttata, C. decemguttata or conspecific eggs, from which surface chemicals were removed by washing them with hexane, was not different from survival on unwashed eggs.