Utilization of dibenzothiophene as sulfur source by <Emphasis Type="Italic">Microbacterium</Emphasis> sp. <Emphasis Type="Italic">NISOC-06</Emphasis>

Oil-polluted soils were sampled from National Iranian South Oil Company (NISOC) for isolation and screening of C–S and not C–C targeted Dibenzothiophene (DBT) degrading microorganisms. Microbacterium sp. NISOC-06, a C–S targeted DBT degrading bacterium, was selected and its desulfurization ability was studied in aqueous phase and water-gasoline biphasic systems. The 16srRNA gene was amplified using universal eubacteria-specific primers, PCR product was sequenced and the sequence of nearly 1,500 bp 16srDNA was studied. Based on Gas Chromatography results Microbacterium sp. NISOC-06 utilized 94.8% of 1 mM DBT during the 2 weeks of incubation. UV Spectrophotometry and biomass production measurements showed that the Microbacterium sp. NISOC-06 was not able to utilize DBT as a carbon source. There was no accumulation of phenolic compounds as Gibb’s assay showed. Biomass production in a biphasic system for which DBT-enriched gasoline was used as the sulfur source indicated the capability of Microbacterium sp. NISOC-06 to desulfurize gasoline.     

Synthesis of orthogonally protected (<Emphasis Type="Italic">3R</Emphasis>,<Emphasis Type="Italic">4S</Emphasis>)- and (<Emphasis Type="Italic">3S</Emphasis>,<Emphasis Type="Italic">4S</Emphasis>)-4,5-diamino-3-hydroxypentanoic acids

Summary.  The paper describes two methods of the synthesis of ethyl (3R,4S)- and (3S,4S)-4-[(benzyloxycarbonyl)amino]-5-[(tert-butyloxycarbonyl)amino]-3-hydroxypentanoates, useful for the syntheses of edeine analogs. Differently N-protected (S)-2,3-diaminopropanoic acid was used as a substrate in both procedures. The absolute configuration of newly generated asymmetric carbon atoms C-3 in β-hydroxy-γ,δ-diamino products was assigned by means of ¹H NMR spectroscopy after their transformation into corresponding piperidin-2-ones. Received May 24, 2002 Accepted October 10, 2002 Published online December 18, 2002 Acknowledgment The authors are indebted to the Faculty of Chemistry, Technical University of Gdańsk for financial support. Authors' address: Zbigniew Czajgucki, M. Sc., Department of Pharmaceutical Technology and Biochemistry, Faculty of Chemistry, Technical University of Gdańsk, 11/12 Narutowicza St., 80-952 Gdańsk, Poland, Fax +48 58 347 11 44, E-mail: zmczaj@wp.pl     

<Emphasis Type="Italic">PVAS3</Emphasis>, a class-II ubiquitous asparagine synthetase from the common bean (<Emphasis Type="Italic">Phaseolus vulgaris)</Emphasis>

A gene encoding a putative asparagine synthetase (AS; EC 6.3.5.4) has been isolated from common bean (Phaseolus vulgaris). A 2.4 kb cDNA clone of this gene (PVAS3) encodes a protein of 570 amino acids with a predicted molecular mass of 64,678 Da, an isoelectric point of 6.45, and a net charge of −5.9 at pH 7.0. The PVAS3 protein sequence conserves all the amino acid residues that are essential for glutamine-dependent AS, and PVAS3 complemented an E. coli asparagine auxotroph, that demonstrates that it encodes a glutamine-dependent AS. PVAS3 displayed significant similarity to other AS. It showed the highest similarity to soybean SAS3 (92.9% identity), rice AS (73.7% identity), Arabidopsis ASN2 (73.2%) and sunflower HAS2 (72.9%). A phylogenetic analysis revealed that PVAS3 belongs to class-II asparagine synthetases. Expression analysis by real-time RT-PCR revealed that PVAS3 is expressed ubiquitously and is not repressed by light.     

High frequency <Emphasis Type="Italic">in vitro</Emphasis> propagation of <Emphasis Type="Italic">Holarrhena antidysenterica</Emphasis> from nodal buds of mature tree

An in vitro method for propagation of Holarrhena antidysenterica Wall. has been developed using nodal explants from mature trees growing in the field. Irrespective of concentrations and combinations of growth regulators used, the axillary and terminal buds sprouted and elongated when inoculated on Murashige and Skoog (MS) medium. The highest numbers of shoots were formed when sprouted shoots were subcultured from MS basal medium onto MS medium containing 2 mg dm⁻³ N⁶-benzyladenine (BA) and 0.5 mg dm⁻³ α-naphthalene acetic acid (NAA). The shoot number further increased upon subculture on MS medium containing 0.5 mg dm⁻³ BA. By repeated sub-culturing of shoots derived from nodal axillary buds, a high frequency multiplication rate was established. The elongated shoots were excised and rooted in auxin free MS basal medium. Ex vitro rooting of in vitro formed shoots was achieved upon dipping the microshoots for 2 min in 2 mg dm⁻³ of indole-3-butyric acid solution. Successful field establishment and high (80–90 %) survival of plants was observed.     

Eradication of <Emphasis Type="Italic">Enterococcus faecalis</Emphasis> by phage therapy in chronic bacterial prostatitis — <Emphasis Type="Italic">case report</Emphasis>

The treatment of three patients suffering from chronic bacterial prostatitis who were qualified for an experimental phage therapy protocol managed at the Phage Therapy Unit in Wrocław is described. They had previously been treated unsuccessfully with long-term targeted antibiotics, autovaccines, and laser biostimulation. Rectal application of phage lysates targeted against Enterococcus faecalis cultured from the prostatic fluid gave encouraging results regarding bacterial eradication, abatement of clinical symptoms of prostatitis, and lack of early disease recurrence.     

Introduction of <Emphasis Type="Italic">OsglyII</Emphasis> gene into <Emphasis Type="Italic">Oryza sativa</Emphasis> for increasing salinity tolerance

Mature seed-derived embryogenic calli of indica rice (Oryza sativa L. cv. PAU201) were induced on semisolid Murashige and Skoog medium supplemented with 2.5 mg dm⁻³ 2,4-dichlorophenoxyacetic acid + 0.5 mg dm⁻³ kinetin + 560 mg dm⁻³ proline + 30 g dm⁻³ sucrose + 8 g dm⁻³ agar. Using OsglyII gene, out of 3180 calli bombarded, 32 plants were regenerated on medium containing hygromycin (30 mg dm⁻³). Histochemical GUS assay of the hygromycin selected calli revealed GUS expression in 50 % calli. Among the regenerants, 46.87 % were GUS positive. PCR analysis confirmed the presence of the transgene of 1 kb in 60 % of independent plants. Further, these plants have been grown to maturity in glasshouse. In vitro screening for salt tolerance showed increase in fresh mass of OsglyII putative transgenic calli (185.4 mg) as compared to control calli (84.2 mg) on 90 mM NaCl after 15 d. When exposed to 150 mM NaCl, OsglyII putative transgenic plantlets showed normal growth while the non-transgenic control plantlets turned yellow and finally did not survive.     

Discovery of <Emphasis Type="Italic">Ophiostoma tsotsi</Emphasis> on <Emphasis Type="Italic">Eucalyptus</Emphasis> wood chips in China

Ophiostoma species such as O. quercus are the most frequent causal agents of sapstain of freshly felled hardwood timber and pulpwood. Many species are regarded as economically important agents of wood degradation. The aim of this study was to identify a collection of Ophiostoma isolates, resembling O. quercus, found on stained Eucalyptus pulpwood chips in China. DNA sequences of the internal transcribed spacer regions, including the 5.8S region, of the ribosomal DNA, and parts of the β-tubulin and elongation factor-1α genes, revealed that the isolates were not O. quercus. Surprisingly, they represented O. tsotsi, a wound-infesting fungus recently described from hardwoods in Africa. In addition, sequence data from an isolate from agarwood in Vietnam, identified in a previous study as belonging to an unknown Pesotum species, were also shown to represent O. tsotsi. A high level of genetic variability was observed among isolates of both O. quercus and O. tsotsi. This was unexpected and suggests that both species have been present in Asia for a significant amount of time.     

Chemical composition of the essential oil from two wormwood species <Emphasis Type="Italic">Artemisia frigida</Emphasis> and <Emphasis Type="Italic">Artemisia argyrophylla</Emphasis>

The composition of the essential oil from the wormwood sage (Artemisia frigida Willd., Asteraceae) of populations growing in the Altai Territory, the Altai Republic, the Khakass Republic, the Tuva Republic, and the East-Kazakhstan region of the Republic of Kazakhstan and the representative species of the silver-leaved wormwood Artemisia argyrophylla Ledeb. growing in the Republic Altai has been studied by chromato-mass spectrometry. An analysis of 15 samples of the essential oil from A. frigida obtained over a period from 1999 to 2007 indicates that samples from different populations have similar sets of the main components: α-pinene (0.2–7.8%), camphene (1.9–5.8%), 1,8-cineole (8.9–33.8%), camphor (6.7–40.0%), borneol (3.9–12.3%), terpine-4-ol (1.5–6.5%), bornyl acetate (1.4–22.0%), and germacrene D (1.4–14.6%). Some samples contain substantial amounts of α- and β-thujones (in total up to 19.1%), which are completely absent in other samples. Some samples contain santolina alcohol (up to 13.8%) and its acetate (up to 4.8%). As differentiated from A. frigida, the essential oil of A. argyrophylla contains yomogi alcohol (1.2%), artemisia ketone (12.9%), artemisia alcohol (3.1%), artemisia alcohol acetate (3.9%), and small amounts of camphor (3.2%), borneol (0.3%), and bornyl acetate (0.2%).     

Response to different oxidants of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis><Emphasis Type="Italic">ure2Δ</Emphasis> mutant

Growth of Saccharomyces cerevisiae ure2Δ mutant strain was investigated in the presence of diverse oxidant compounds. The inability of the strain to grow on a medium supplemented with H₂O₂ was confirmed and a relationship between diminishing levels of glutathione (GSH) and peroxide sensitivity was established. Data for the lack of significant effect of URE2 disruption on the cellular growth in the presence of paraquat and menadione were obtained. The possible role of Ure2p in acquiring sensitivity to oxidative stress by means of its regulatory role in the GATA signal transduction pathway was discussed. It was suggested that the susceptibility of ure2Δ mutant to the exogenous hydrogen peroxide can result from increased GSH degradation due to the deregulated localization of the γ-glutamyl transpeptidase activating factors Gln3/Gat1. The important role of Ure2p in in vivo glutathione-mediated reactive oxygen species (ROS) scavenging was shown by measuring the activity of antioxidant enzymes glutathione peroxidase, superoxide dismutase (SOD) and catalase in an URE2 disrupted strain. A time-dependent increase in SOD and catalase activity was observed. More importantly, it was shown that the ure2 mutation could cause significant disturbance in cellular oxidant balance and increased ROS level.     

Morphological and functional abnormalities in neuromuscular junctions of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> induced by human <Emphasis Type="Italic">APP</Emphasis> gene expression

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by a loss of neurocortical and hippocampal synapses that precedes amyloidosis and neurodegeneration and closely correlates with memory impairment. Mutations in the amyloid precursor protein (APP) cause familial AD and result in increased production of amyloid-β-protein (Aβ). To gain insight into the synaptic effects of APP protein in AD patients, wild-type APP, its mutant form APP-Swedish responsible for familial AD, and human beta-secretase gene were expressed in motor neurons of Drosophila melanogaster larvae. It was found that targeted expression of APP (APP-Swedish) in Drosophila larval motor neurons caused significant morphological and functional changes in neuromuscular junctions (NMJs)-a dramatic increase in the number of synaptic boutons and altered exocytosis revealed by incorporation of the styryl dye FM4-64. Analysis of the number and distribution of mitochondria showed that motor neurons overexpressing APP (APP-Swedish) had a significant reduction of functional mitochondria in the presynaptic terminal. Significant synaptic abnormalities were observed with APP (APP-Swedish) expression, as well as for double transgenes bearing APP (APP-Swedish) and human beta-secretase (BACE), which caused secretion of amyloid beta protein (Aβ). We suggest that APP participates in regulation of synaptic functions and its elevated expression leads to synaptic pathology independently from Aβ neurotoxic effects.     

Presence of <Emphasis Type="Italic">C. albidus</Emphasis>, <Emphasis Type="Italic">C. laurentii</Emphasis> and <Emphasis Type="Italic">C. uniguttulatus</Emphasis> in Crop and Droppings of Pigeon Lofts (<Emphasis Type="Italic">Columba livia</Emphasis>)

Columba livia is an important reservoir and carrier of Cryptococcus neoformans, Cryptococcus uniguttulatus, Cryptococcus laurentii and Cryptococcus albidus. Upper digestive tract of this species is also known as a habitat for Cryptococcus neoformans. Given the increasing clinical interest of this microorganism, 331 swabs from crop and 174 dropping samples from pigeon lofts in Grand Canary Island have been studied. The obtained results show an extensive presence samples 81 positive (24.47%) of Cryptococcus spp. in analysed crops: 32 (9.66%) for C. neoformans, 24 (7.2%) for C. uniguttulatus, 23 (6.9%) for C. albidus and 2 (0.6%) for C. laurentii. In the same way, Cryptococcus spp was also isolated in 82 (47.13%), dropping samples: C. neoformans in 59 (33.9%), C. uniguttulatus, in 9 (5.17%), C. laurentii in 8 (4.59%) and C. albidus in 6 (3.44%) of the investigated samples, respectively. The cryptococcosis produced by species of cryptococci other than C. neoformans has become more important during the last decade, supporting the study on the role of pigeon in the epidemiology of this disease.     

Target site selection by the <Emphasis Type="Italic">mariner</Emphasis>-like element, <Emphasis Type="Italic">Mos1</Emphasis>

The eukaryotic transposon Mos1 is a class-II transposable element that moves using a “cut-and-paste” mechanism in which the transposase is the only protein factor required. The formation of the excision complex is well documented, but the integration step has so far received less investigation. Like all mariner-like elements, Mos1 was thought to integrate into a TA dinucleotide without displaying any other target selection preferences. We set out to synthesize what is currently known about Mos1 insertion sites, and to define the characteristics of Mos1 insertion sequences in vitro and in vivo. Statistical analysis can be used to identify the TA dinucleotides that are non-randomly targeted for transposon integration. In vitro, no specific feature determining target choice other than the requirement for a TA dinucleotide has been identified. In vivo, data were obtained from two previously reported integration hotspots: the bacterial cat gene and the Caenorhabditis elegans rDNA locus. Analysis of these insertion sites revealed a preference for TA dinucleotides that are included in TATA or TA × TA motifs, or located within AT-rich regions. Analysis of the physical properties of sequences obtained in vitro and in vivo do not help to explain Mos1 integration preferences, suggesting that other characteristics must be involved in Mos1 target choice.     

An endophytic <Emphasis Type="Italic">Neurospora</Emphasis> sp. from <Emphasis Type="Italic">Nothapodytes foetida</Emphasis> producing camptothecin

The medicinal plant, Nothapodytes foetida, contains a number of important alkaloids like camptothecin (an anticancer drug molecule), but its concentration is less to meet the existing demand of this important molecule, in an effort for accessible availability of camptothecin. An endophyte (designated ZP5SE) was isolated from the seed of Nothapodytes foetida and was examined as a potential source of anticancer drug lead compound, i.e., camptothecin, when grown in Sabouraud liquid culture media under shake flask conditions. The presence of an anticancer compound (camptothecin) in this fungus was confirmed by chromatographic and spectroscopic methods in comparison with authentic camptothecin. Isolated endophyte (Neurospora crassa) producing camptothecin may become an easily accessible source for the production of a precursor anticancer drug molecule in the future at a large scale. Published in Russian in Prikladnaya Biokhimiya i Mikrobiologiya, 2008, Vol. 44, No. 2, pp. 225–231. The text was submitted by the authors in English.     

Expression of Two Self-enhancing Antifreeze Proteins from the Beetle <Emphasis Type="Italic">Dendroides canadensis</Emphasis> in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Antifreeze proteins depress the non-equilibrium freezing point of aqueous solutions, but only have a small effect on the equilibrium melting point. This difference between the freezing and melting points has been termed thermal hysteresis activity (THA). THA identifies the presence and relative activity of antifreeze proteins. Two antifreeze protein cDNAs, dafp-1 and dafp-4, encoding two self-enhancing (have a synergistic effect on THA) antifreeze proteins (DAFPs) from the beetle Dendroides canadensis, were introduced into the genome of Arabidopsis thaliana via Agrobacterium-mediated floral dip transformation. Southern blot analysis indicated multiple insertions of transgenes. Both DAFP-1 and/or DAFP-4 were expressed in transgenic A. thaliana as shown by RT-PCR and Western blot. Apoplastic fluid from T ₃ DAFP-1 + DAFP-4-producing transgenic A. thaliana exhibited THA in the range of 1.2–1.35°C (using the capillary method to determine THA), demonstrating the presence of functioning antifreeze proteins (with signal peptides for extracellular secretion). The freezing temperature of DAFP-1 + DAFP-4-producing transgenic A. thaliana was lowered by approximately 2–3°C compared with the wild type.     

Ectopic expression of two MADS box genes from orchid (<Emphasis Type="Italic">Oncidium</Emphasis> Gower Ramsey) and lily (<Emphasis Type="Italic">Lilium longiflorum)</Emphasis> alters flower transition and formation in <Emphasis Type="Italic">Eustoma grandiflorum</Emphasis>

Lisianthus [Eustoma grandiflorum (Raf.) Shinn] is a popular cut flower crop throughout the world, and the demand for this plant for cut flowers and potted plants has been increasing worldwide. Recent advances in genetic engineering have enabled the transformation and regeneration of plants to become a powerful tool for improvement of lisianthus. We have established a highly efficient plant regeneration system and Agrobacterium-mediated genetic transformation of E. grandiflorum. The greatest shoot regeneration frequency and number of shoot buds per explant are observed on media supplemented with 6-Benzylaminopurine (BAP) and α-Naphthalene acetic acid (NAA). We report an efficient plant regeneration system using leaf explants via organogenesis with high efficiency of transgenic plants (15%) in culture of 11 weeks’ duration. Further ectopic expression of two MADS box genes, LMADS1-M from lily (Lilium longiflorum) and OMADS1 from orchid (Oncidium Gower Ramsey), was performed in E. grandiflorum. Conversion of second whorl petals into sepal-like structures and alteration of third whorl stamen formation were observed in the transgenic E. grandiflorum plants ectopically expressing 35S::LMADS1-M. 35S::OMADS1 transgenic E. grandiflorum plants flowered significantly earlier than non-transgenic plants. This is the first report on the ectopic expression of two MADS box genes in E. grandiflorum using a simple and highly efficient gene transfer protocol. Our results reveal the potential for floral modification in E. grandiflorum through genetic transformation.     

Activity-density of <Emphasis Type="Italic">Pardosa cribata</Emphasis> in Spanish citrus orchards and its predatory capacity on <Emphasis Type="Italic">Ceratitis capitata</Emphasis> and <Emphasis Type="Italic">Myzus persicae</Emphasis>

The wolf spider Pardosa cribata Simon is the most abundant ground-dwelling spider inhabiting citrus orchards in eastern Spain. However, little is known about its activity-density and its predatory role in the citrus agrosystem. Here we report on the activity-density of P. cribata monitored by pitfall traps, and on its capacity to prey on two citrus pests that appear both in the citrus canopy and the ground cover, Ceratitis capitata (Wiedemman) and Myzus persicae (Sulzer), respectively. Pardosa cribata was present in citrus orchards throughout the year, with a peak in spring and a higher peak in summer. Pardosa cribata preyed on adults and third-instar larvae but not on pupae of C. capitata. A type II functional response was obtained for teneral-like adults, with an estimated attack rate (a′) of 0.771 ± 0.213 days⁻¹ and a handling time (T _h) of 0.051 ± 0.013 days. Pardosa cribata also preyed efficiently on M. persicae, giving a type II functional response with an estimated attack rate and handling time of 2.833 ± 0.578 days⁻¹ and 0.031 ± 0.001 days, respectively. The data reported here indicate that this wolf spider could play an important role in regulating both these pests, and therefore might contribute to developing conservation biological control strategies for citrus pests. Handling Editor: Arne Jenssen.     

Development of a real-time TaqMan assay to detect <Emphasis Type="Italic">mendocina</Emphasis> sublineage <Emphasis Type="Italic">Pseudomonas</Emphasis> species in contaminated metalworking fluids

A TaqMan quantitative real-time polymerase chain reaction (qPCR) assay was developed for the detection and enumeration of three Pseudomonas species belonging to the mendocina sublineage (P. oleovorans, P. pseudoalcaligenes, and P. oleovorans subsp. lubricantis) found in contaminated metalworking fluids (MWFs). These microbes are the primary colonizers and serve as indicator organisms of biodegradation of used MWFs. Molecular techniques such as qPCR are preferred for the detection of these microbes since they grow poorly on typical growth media such as R2A agar and Pseudomonas isolation agar (PIA). Traditional culturing techniques not only underestimate the actual distribution of these bacteria but are also time-consuming. The primer–probe pair developed from gyrase B (gyrB) sequences of the targeted bacteria was highly sensitive and specific for the three species. qPCR was performed with both whole cell and genomic DNA to confirm the specificity and sensitivity of the assay. The sensitivity of the assay was 10¹ colony forming units (CFU)/ml for whole cell and 13.7 fg with genomic DNA. The primer–probe pair was successful in determining concentrations from used MWF samples, indicating levels between 2.9 × 10³ and 3.9 × 10⁶ CFU/ml. In contrast, the total count of Pseudomonas sp. recovered on PIA was in the range of <1.0 × 10¹ to 1.4 × 10⁵ CFU/ml for the same samples. Based on these results from the qPCR assay, the designed TaqMan primer–probe pair can be efficiently used for rapid (within 2 h) determination of the distribution of these species of Pseudomonas in contaminated MWFs.     

Mapping species differences for adventitious rooting in a <Emphasis Type="Italic">Corymbia torelliana</Emphasis> × <Emphasis Type="Italic">Corymbia citriodora</Emphasis> subspecies <Emphasis Type="Italic">variegata</Emphasis> hybrid

Quantitative trait loci (QTL) detection was carried out for adventitious rooting and associated propagation traits in a second-generation outbred Corymbia torelliana × Corymbia citriodora subspecies variegata hybrid family (n = 186). The parental species of this cross are divergent in their capacity to develop roots adventitiously on stem cuttings and their propensity to form lignotubers. For the ten traits studied, there was one or two QTL detected, with some QTL explaining large amounts of phenotypic variation (e.g. 66% for one QTL for percentage rooting), suggesting that major effects influence rooting in this cross. Collocation of QTL for many strongly genetically correlated rooting traits to a single region on linkage group 12 suggested pleiotropy. A three locus model was most parsimonious for linkage group 12, however, as differences in QTL position and lower genetic correlations suggested separate loci for each of the traits of shoot production and root initiation. Species differences were thought to be the major source of phenotypic variation for some rooting rate and root quality traits because of the major QTL effects and up to 59-fold larger homospecific deviations (attributed to species differences) relative to heterospecific deviations (attributed to standing variation within species) evident at some QTL for these traits. A large homospecific/heterospecific ratio at major QTL suggested that the gene action evident in one cross may be indicative of gene action more broadly in hybrids between these species for some traits.