An endophytic Neurospora sp. from Nothapodytes foetida 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, so in an effort for accessible availability of camptothecin. An endophyte (designated ZP5SE) was isolated from the seed of Nothapodytes foetida and was examined as potential source of anticancer drug lead compound i.e. camptothecin, when grown in Sabouraud liquid culture media under shake flask conditions. The presence of 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 precursor anticancer drug molecule in future at large scale.     

<Emphasis Type="Italic">In vitro</Emphasis> cytotoxicity of an endophytic fungus isolated from<Emphasis Type="Italic">Nothapodytes foetida</Emphasis>

An attempt has been made to assessin vitro cytotoxicity of an endophytic fungus fromNothapodytes foetida. Various human cancer cell lines (liver HEP-2, lung A-549, ovary OVAR-5, prostate PC-3, cervix Hela, colon HCT-15, oral cell line KB, CNS SNB-78, were used.In vitro cytotoxicity of camptothecin (CPT) isolated from the fungus was done where OVAR-5 cell line showed maximum inhibition and HEP-2 cell line was least sensitive with this compound.In vitro cytotoxicity of fractions/extracts from endophyte was carried out where ethyl acetate fraction showed sufficient growth inhibition against all the cell lines.     

Somatic embryogenesis,plant regeneration,and the evaluation of camptothecin content in <Emphasis Type="Italic">Nothapodytes foetida</Emphasis>

Summary Somatic embryogenesis and plant regeneration have been achieved in Nothapodytes foetida, which is known for its rich source of anti-cancer and anti-AIDS alkaloids. Callus cultures were initiated from immature zygotic embryos cultured on Murashige and Skoog's (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid, 6-benzyladenine (BA), and kinetin. MS medium devoid of plant growth regulators favored the development of globular somatic embryos that differentiated further into plantlets. Plantlet regeneration efficiency was effectively increased on MS medium supplemented with BA. Over 90% of the in vitro plantlets survived when transferred to the soil. Alkaloids were detected in different stages of somatic embryos, regenerated plantlets, and different parts of the 2-yr-old regenerated plants. The somatic embryos contains camptothecin (0.011% dry weight. DW) and 9-methoxycamptothecin (0.0028% DW). Two-yearold field-grown plants obtained from somatic embryos were analyzed and contained higher levels of camptothecin (0.20% DW) and 9-methoxycamptothecin. (0.097% DW) accumulated in roots, followed by stem and leaves. Alkaloids were quantified and identified by TLC and HPLC.     

Multiple host-ant use by the predatory social parasite <Emphasis Type="Italic">Phengaris</Emphasis> (=<Emphasis Type="Italic">Maculinea</Emphasis>) <Emphasis Type="Italic">arion</Emphasis> (Lepidoptera,Lycaenidae)

Phengaris (=Maculinea) arion is an endangered social parasite of Myrmica ants, and for a very long time was considered as specific to Myrmica sabuleti. Previous studies carried out in Poland suggested some discrepancies within this assumption, and therefore a much more intensive survey was undertaken. The host ant use of P. arion was studied at five sites in different types of biotopes in Poland, i.e. xerothermal grasslands where Thymus pulegioides was used as a larval food plant by the butterfly, and more or less sandy biotopes with Thymus serpyllum. Altogether nine Myrmica species were recorded, and considerable variation in species composition and density of nests was recorded. At four localities M. sabuleti proved to be the most common ant. A total of 529 Myrmica nests were examined, and only 20 of them contained larvae and pupae of P. arion. Host ants belonged to five different species, i.e. M. sabuleti, Myrmica scabrinodis, Myrmica schencki, Myrmica lobicornis and Myrmica hellenica. Only at one site (NE Poland) was a significant heterogeneity in parasitation rates among Myrmica species detected. M. lobicornis was the most often infested ant there, which may suggest local specialisation of the butterfly. Overall low parasitism rates may explain the vulnerability of P. arion in Central Europe but further studies are also necessary.     

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.     

High-level expression of <Emphasis Type="Italic">Camelid</Emphasis> nanobodies in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis>

Nanobodies (or VHHs) are single-domain antigen-binding fragments derived from Camelid heavy chain-only antibodies. Their small size, monomeric behaviour, high stability and solubility, and ability to bind epitopes not accessible to conventional antibodies make them especially suitable for many therapeutic and biotechnological applications. Here we describe high-level expression, in Nicotiana benthamiana, of three versions of an anti-hen egg white lysozyme (HEWL) nanobody which include the original VHH from an immunized library (cAbLys3), a codon-optimized derivative, and a codon-optimized hybrid nanobody comprising the CDRs of cAbLys3 grafted onto an alternative ‘universal’ nanobody framework. His6- and StrepII-tagged derivatives of each nanobody were targeted for accumulation in the cytoplasm, chloroplast and apoplast using different pre-sequences. When targeted to the apoplast, intact functional nanobodies accumulated at an exceptionally high level (up to 30% total leaf protein), demonstrating the great potential of plants as a nanobody production system.     

Antinociceptive Effect of <Emphasis Type="Italic">Salvia</Emphasis> Extract on Cisplatin-Induced Hyperalgesia in Mice

Experiments carried out on mice demonstrated that administration of a platinum-based drug, cisplatin, extensively used in anticancer chemotherapy, exerts significant hyperalgesic effects; it intensifies both phases of pain behavioral reactions induced in the formalin test. When introduction of cisplatin was combined with i.p. injections of 100 mg/kg of an aqueous-alcoholic extract from the leaves of Salvia officinalis, the second phase of cisplatin-enhanced pain in the formalin test was effectively suppressed; the effect was comparable with that provided by injections of morphine or even more intense.     

<Emphasis Type="Italic">Bacillus anthracis</Emphasis>, <Emphasis Type="Italic">Francisella tularensis</Emphasis> and <Emphasis Type="Italic">Yersinia pestis</Emphasis>. The most important bacterial warfare agents — <Emphasis Type="Italic">review</Emphasis>

There are three most important bacterial causative agents of serious infections that could be misused for warfare purposes: Bacillus anthracis (the causative agent of anthrax) is the most frequently mentioned one; however, Fracisella tularensis (causing tularemia) and Yersinia pestis (the causative agent of plague) are further bacterial agents enlisted by Centers for Disease Control and Prevention into the category A of potential biological weapons. This review intends to summarize basic information about these bacterial agents. Military aspects of their pathogenesis and the detection techniques suitable for field use are discussed.     

Comparison of the Antioxidant Properties and the Toxicity of <Emphasis Type="Italic">p</Emphasis>,<Emphasis Type="Italic">p</Emphasis>′-Dichlorodiphenyl Ditelluride with the Parent Compound,Diphenyl Ditelluride

The hypothesis to be tested in this study is whether the introduction of the chloro group into diphenyl ditelluride molecule (p,p′-dichlorodiphenyl ditelluride, compound 1b) alters the antioxidant and scavenging activity of diphenyl ditelluride (compound 1a) in vitro. The results revealed that 1a and 1b had a potent antioxidant activity in vitro. However, the introduction of a functional group, chloro, into diphenyl ditelluride molecule (1b) did not cause great alterations in the antioxidant action of diphenyl ditelluride against lipid peroxidation, protein carbonyl, and scavenging of 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate) and 2,2′-diphenyl-1-picrylhydrazyl radicals. Based on the in vitro results, different doses (0.25 and 0.75 μmol/kg) of 1a and 1b or vehicle (canola oil, 1 ml/kg) were administered to rats to investigate if the presence of chloro into diphenyl ditelluride molecule reduces its toxicity. The data demonstrate that the chloro group introduced into diphenyl ditelluride molecule did not alter the acute oral toxicity in rats. The administration of compound 1a in rats only altered the urea level, while compound 1b caused alterations in all toxicological parameters analyzed (alanine aminotransferase and aspartate aminotransferase activities, urea and creatinine levels) in plasma of rats. The results of the present investigation support similar antioxidant and scavenging activities of 1a and 1b in rat liver homogenate in vitro. Furthermore, the presence of chloro into diphenyl ditelluride molecule did not alter the mortality index but increased toxicity of diphenyl ditelluride in rats.     

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.     

RETRACTED ARTICLE: The AtCCX1 transporter mediates salinity tolerance in both <Emphasis Type="Italic">Arabidopsis</Emphasis> and yeast

The only species in the genus Passiflora (Passifloraceae) known to produce resin glands is P. foetida. These glands are secretory trichomes mainly present on the floral bracts and leaf stipules. The secretion produced by these glands has received attention recently due to the presence of substances with pharmacological properties. Attempts to apply in vitro cell culture methods for the large scale production of highly valuable metabolites has been rather limited due to the fact that these compounds are produced by highly differentiated secretory cells in trichomes which are seldom obtained or because differentiation is inhibited by in vitro conditions. Here we describe the in vitro plant regeneration of P. foetida obtained via organogenesis, using mature zygotic embryos as explants. Differentiated plantlets and, more important, the de novo differentiation of secretory trichomes in vitro could be observed in less than 30 days. There was a clear effect of the concentration of 2,4-dichlorophenoxyacetic acid in the culture media on the regeneration of plants and on the differentiation of glandular trichomes. Our results should be useful for the micropropagation of P. foetida, as well as for studies of the process of secretory trichome differentiation and the implemention of biotechnological methodologies for in vitro mass production of passifloricin and/or other substances present in the P. foetida resin.     

Comparative analysis of <Emphasis Type="Italic">iol</Emphasis> clusters in <Emphasis Type="Italic">Lactobacillus casei</Emphasis> strains

The present study was designed to expand genetic knowledge of myo -inositol (MI) metabolism in Lactobacillus casei. Twenty-four L. casei isolates of dairy origin were tested for the presence of iol cluster. PCR screening revealed eight strains encoded functions involved in MI utilization, of which one strain was able to use MI as carbon source. To gain a deeper understanding of the function of iol genes, four of the eight observed iol clusters were subjected to the full sequencing procedure. The results showed that the iol cluster was not a common feature among dairy L. casei strains. In addition, the four iol clusters were highly similar to one another in terms of sequence similarity and operon architecture. However, abundant polymorphisms that comprised a majority of synonymous mutations were detected throughout the full sequences. Three of them distributed among iolB, iolC, and iolT genes were found in linkage to MI-negative phenotype. Compared with other bacterial iol clusters, the iol cluster of L. casei showed a high similarity with that of Bacillus subtilis.     

Effects of <Emphasis Type="Italic">crp</Emphasis> deletion in <Emphasis Type="Italic">Salmonella enterica</Emphasis> serotype Gallinarum

Background  

Salmonella enterica serotype Gallinarum (S. Gallinarum) remains an important pathogen of poultry, especially in developing countries. There is a need to develop effective and safe vaccines. In the current study, the effect of crp deletion was investigated with respect to virulence and biochemical properties and the possible use of a deletion mutant as vaccine candidate was preliminarily tested.     

Magnetic field perception in the Rainbow Trout, <Emphasis Type="Italic">Oncorhynchus</Emphasis> <Emphasis Type="Italic">mykiss</Emphasis>

In this study, we present evidence for the perception of different magnetic field parameters in a facultative anadromous fish species of the family Salmonidae. Magnetic field perception of the rainbow trout, Oncorhynchus mykiss, was demonstrated with a heartbeat conditioning test. The electrocardiogram was measured with subcutaneously inserted silver wire electrodes in freely swimming fish. We demonstrate a conditioned response (i.e. a significant longer interval between two heartbeats) to an intensity/inclination shift for three adult and two juvenile rainbow trouts. Moreover, a conditioned response to a 90° direction shift was demonstrated for three adult and two juvenile trouts. These findings support the hypothesis that the rainbow trout is able to perceive different magnetic field parameters. Furthermore, the study demonstrates magnetosensation in different developmental stages in the rainbow trout, i.e. juvenile and adult fish.     

Fitness aspects of transgenic <Emphasis Type="Italic">Aedes fluviatilis</Emphasis> mosquitoes expressing a <Emphasis Type="Italic">Plasmodium</Emphasis>-blocking molecule

Vector-born diseases cause millions of deaths every year globally. Alternatives for the control of diseases such as malaria and dengue fever are urgently needed and the use of transgenic mosquitoes that block parasite/virus is a sound strategy to be used within control programs. However, prior to use transgenic mosquitoes as control tools, it is important to study their fitness since different biological aspects might influence their ability to disseminate and compete with wild populations. We previously reported the construction of four transgenic Aedes fluviatilis mosquito lines expressing a Plasmodium- blocking molecule (mutated bee venom phospholipase A₂–mPLA₂). Presently we studied two aspects of their fitness: body size, that has been used as a fitness-related status, and the expression of major enzymes classes involved in the metabolism of xenobiotics, including insecticides. Body size analysis (recorded by geometric wing morphometrics) indicated that both male and female mosquitoes were larger than the non-transgenic counterparts, suggesting that this characteristic might have an impact on their overall fitness. By contrast, no significant difference in the activity of enzymes related to metabolic insecticide resistance was detected in transgenic mosquitoes. The implication on fitness advantage of these features, towards the implementation of this strategy, is further discussed.     

<Emphasis Type="Italic">In vitro</Emphasis> regenerating plantlets in <Emphasis Type="Italic">Pandanus amaryllifolius</Emphasis> Roxb. as a model system to study the development of lower epidermal papillae

Pandanus amaryllifolius Roxb. has been identified as a rich source of principal basmati aroma compound 2 acetyl-1-pyrroline (2AP). An easy and efficient protocol for clonal propagation of P. amaryllifolius Roxb. using lateral buds as an explant has been developed and the in vitro-raised seedlings were used to study the lower epidermal papillae development. The principal aroma compound 2AP along with other aroma compounds are stored in the lower epidermis papillae of leaf. The developmental pattern of these papillae was traced out using scanning electron microscopy. It was observed that stomata act as the site of initiation for development of the papillae followed by their lateral spread across the epidermal cells. During development, the first papillae bulged out as a protrusion of the lower epidermis that further metamorphosed into well-grown papillae. These developments are well-correlated with 2AP contents, in which the in vitro-raised seedlings had less 2AP contents (66.99 ppb) than the mother plant (96.88 ppb).     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of the plant pathogenic fungus <Emphasis Type="Italic">Rosellinia necatrix</Emphasis>

Rosellinia necatrix is a soil-borne root pathogen affecting a wide range of commercially important plant species. The mycelium of R. necatrix was transformed to hygromycin B resistance by an Agrobacterium tumefaciens-mediated transformation system using a binary plasmid vector containing the hygromycin B phosphotransferase (hph) gene controlled by the heterologous fungal Aspergillus nidulans P-gpd (glyceraldehyde 3-phosphate dehydrogenase) promoter and the trpC terminator. Co-cultivation of R. necatrix strain W1015 and A. tumefaciens strain AGL-1 at 25°C using the binary vector pAN26-CB1300, which contained the hygromycin B resistance cassette based on pAN26 and pCAMBIA1300, resulted in high frequencies of transformation. The presence of the hph gene in the transformants was detected by PCR, and single-copy integration of the marker gene was demonstrated by Southern b lot analy s is. This report of an Agrobacterium-mediated transformation method should allow the development of T-DNA tagging as a system for insertional mutagenesis in R. necatrix and provide a simple and reliable method for genetic manipulation.     

Methylglyoxal destroys <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> crown gall tumours in <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> without any adverse effect on the host plant

Methylglyoxal (MG) is a highly reactive α-oxoaldehyde, demonstrating anticancer effect on plant neoplastic tumours. In in vivo studies it was observed that MG destroyed crown gall tumours in Nicotiana tabacum produced by Agrobacterium tumefaciens, without any adverse effect on the host. The efficacy of MG in comparison to other anticancer drugs viz. cisplatin and ellagic acid in the treatment of crown gall was investigated. A slight degeneration of galls was noted in plants treated with cisplatin and ellagic acid but the plants died subsequently. With MG however, crown galls were completely cured and the plants completed their usual life cycle by flowering and producing seeds. MG inhibited the respiration of crown gall calluses suggesting that energy depletion resulted in tumour destruction.     

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.