<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

Characterization of the new human pleomorphic undifferentiated sarcoma <Emphasis Type="Italic">TP53</Emphasis>-<Emphasis Type="Italic">null</Emphasis> cell line <Emphasis Type="Italic">mfh</Emphasis>-<Emphasis Type="Italic">val2</Emphasis>

Pleomorphic undifferentiated sarcoma (PUS), also called malignant fibrous histiocytoma, is a soft tissue sarcoma which occurs predominantly in the extremities. Its origin is a poorly defined mesenchymal cell, which derives to histiocytic and fibroblastic cells. The patient, a 58 year-old man, presented a lesion located in the forearm composed by spindle cells and multinucleated giant cells, which expressed vimentin and adopted a histological pattern formed by irregular-swirling fascicles. Cells were cultured in vitro and a new cell line was established. We characterized this new cell line by histological analyses, cytogenetics (using G-bands and spectral karyotype technique) and cytometric analyses. Cells were grown in culture for more than 100 passages. They had elongated or polygonal morphology. The cells presented a saturation rate of 70,980 cells/cm², a plating efficiency of 21.5% and a mitotic index of 21 mitoses per field. The cell line was tumorigenic in nude mice. The ploidy study using flow cytometry revealed an aneuploid peak with a DNA index of 1.43. A side population was detected, demonstrating the presence of stem and progenitor cells. Cytogenetics showed a hypotriploid range with many clonal unbalanced rearrangements. Loss of p53 gene was evidenced by MLPA. We describe, for the first time, the characterization of a new human PUS TP53-null cell line called mfh-val2. Mfh-val2 presents a wide number of applications as a TP53-null cell line and a great interest in order to characterize genetic alterations influencing the oncogenesis or progression of PUS and to advance in the biological investigation of this tumor.     

Expression of <Emphasis Type="Italic">recA</Emphasis> gene of <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis> cells

Plasmids pKS5 and pKSrec30 carrying normal and mutant alleles of the Deinococcus recA gene controlled by the lactose promoter slightly increase radioresistance of Escherichia coli cells with mutations in genes recA and ssb. The RecA protein of D. radiodurans is expressed in E. coli cells, and its synthesis can be supplementary induced. The radioprotective effect of the xenologic protein does not exceed 1.5 fold and yields essentially to the contribution of plasmid pUC19-recA1.1 harboring the E. coli recA ⁺ gene in the recovery of resistance of the ΔrecA deletion mutant. These data suggest that the expression of D. radiodurans recA gene in E. coli cells does not complement mutations at gene recA in the chromosome possibly due to structural and functional peculiarities of the D. radiodurans RecA protein.     

Study of tauopathies by comparing <Emphasis Type="Italic">Drosophila</Emphasis> and human <Emphasis Type="Italic">tau</Emphasis> in <Emphasis Type="Italic">Drosophila</Emphasis>

The microtubule-binding protein tau has been investigated for its contribution to various neurodegenerative disorders. However, the findings from transgenic studies, using the same tau transgene, vary widely among different laboratories. Here, we have investigated the potential mechanisms underlying tauopathies by comparing Drosophila (d-tau) and human (h-tau) tau in a Drosophila model. Overexpression of a single copy of either tau isoform in the retina results in a similar rough eye phenotype. However, co-expression of Par-1 with d-tau leads to lethality, whereas co-expression of Par-1 with h-tau has little effect on the rough eye phenotype. We have found analogous results by comparing larval proteomes. Through genetic screening and proteomic analysis, we have identified some important potential modifiers and tau-associated proteins. These results suggest that the two tau genes differ significantly. This comparison between species-specific isoforms may help to clarify whether the homologous tau genes are conserved. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. This study was supported by the National Science Foundation of China (30270341; 30630028), the Multidisciplinary Program (Brain and Mind) of the Chinese Academy of Sciences, the Major State Basic Research Program (“973 program”; G2000077800; G2006CB806600; 2006CB911003), the Precedent Project of Important Intersectional Disciplines in the Knowledge Innovation Engineering of the Chinese Academy of Sciences (KJCX1-09-03).     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

<Emphasis Type="Italic">KRAS</Emphasis>, <Emphasis Type="Italic">BRAF</Emphasis>, <Emphasis Type="Italic">EGFR</Emphasis> and <Emphasis Type="Italic">HER2</Emphasis> gene status in a Spanish population of colorectal cancer

To evaluate the KRAS, BRAF, EGFR, and HER2 gene status in colorectal cancer by novel techniques and evaluate whether anti-HER2 therapies could be offered in the treatment of these patients. There are conflicting data on the prevalence of BRAF mutations and EGFR and HER2 gene amplification in colorectal KRAS wild type patients. In our study we tried to evaluate these expressions and their relationship to future treatment assays. Clinical–pathological data and paraffin-embedded specimens were collected from 186 patients who underwent colorectal resections at General Yagüe Hospital in Burgos, Spain. KRAS and BRAF status was analyzed by real-time PCR in all patients. EGFR and HER2/NEU gene amplification was detected using fluorescent in situ hybridisation technique (FISH) in 38 KRAS and BRAF wild type patients. KRAS mutations were present in 48% of the colorectal cancer patients. BRAF mutations were present in 6.25% of the KRAS wild type patients. EGFR and HER2 gene amplification was observed in 5.3% and 26.3%, respectively, of KRAS and BRAF wild type colorectal cancer patients. HER2, but not EGFR gene amplification, was frequently observed in KRAS and BRAF wild type colorectal cancer patients. These data indicate that HER2 amplification could be one of the genes to be considered in the therapeutic management of colorectal cancer.     

New combinations and typifications in <Emphasis Type="Italic">Bistorta</Emphasis>, <Emphasis Type="Italic">Persicaria</Emphasis>, <Emphasis Type="Italic">Polygonum,</Emphasis> and <Emphasis Type="Italic">Rumex</Emphasis> (Polygonaceae)

New combinations are proposed in anticipation of the Polygonaceae treatment in the forthcoming volume of Intermountain Flora: Polygonum kelloggii var. esotericum, P. kelloggii var. watsonii , Rumex densiflorus var. pycnanthus , R. salicifolius var. utahensis, and R. occidentalis var. tomentellus. Typifications are proposed to facilitate ongoing studies in Polygonaceae and to maintain current usage.     

In planta transformation of <Emphasis Type="Italic">Notocactus scopa</Emphasis> cv. <Emphasis Type="Italic">Soonjung</Emphasis> by <Emphasis Type="Italic">Agrobacterium </Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>

Notocactus scopa cv. Soonjung was subjected to in planta Agrobacterium tumefaciens-mediated transformation with vacuum infiltration, pin-pricking, and a combination of the two methods. The pin-pricking combined with vacuum infiltration (20-30 cmHg for 15 min) resulted in a transformation efficiency of 67-100%, and the expression of the uidA and nptII genes was detected in transformed cactus. The established in planta transformation technique generated a transgenic cactus with higher transformation efficiency, shortened selection process, and stable gene expression via asexual reproduction. All of the results showed that the in planta transformation method utilized in the current study provided an efficient and time-saving procedure for the delivery of genes into the cactus genome, and that this technique can be applied to other asexually reproducing succulent plant species.     

Regulatory activity of heterologous gene-activator <Emphasis Type="Italic">xlnR</Emphasis> of <Emphasis Type="Italic">Aspergillus niger</Emphasis> in <Emphasis Type="Italic">Penicillium canescens</Emphasis>

The gene encoding the xlnR xylanolytic activator of the heterologous fungus Aspergillus niger was incorporated into the Penicillium canescens genome. Integration of the xlnR gene resulted in the increase in a number of activities, i.e. endoxylanase, β-xylosidase, α-L-arabinofuranosidase, α-galactosidase, and feruloyl esterase, compared to the host P. canescens PCA 10 strain, while β-galactosidase, β-glucosidase, endoglucanase, and CMCase activities remained constant. Two different expression constructs were developed. The first consisted of the nucleotide sequence containing the mature P. canescens phytase gene under control of the axhA promoter region gene encoding A. niger (1,4)-β-D-arabinoxylan-arabinofuranohydrolase. The second construct combined the P. canescens phytase gene and the bgaS promoter region encoding homologous β-galactosidase. Both expression cassettes were transformed into P. canescens host strain containing xlnR. Phytase synthesis was observed only for strains with the bgaS promoter on arabinose-containing culture media. In conclusion, the bgaS and axhA promoters were regulated by different inducers and activators in the P. canescens strain containing a structural tandem of the axhA promoter and the gene of the xlnR xylanolytic activator.     

Functional conservation of the human <Emphasis Type="Italic">EXT1</Emphasis> tumor suppressor gene and its <Emphasis Type="Italic">Drosophila</Emphasis> homolog <Emphasis Type="Italic">tout</Emphasis><Emphasis Type="Italic">velu</Emphasis>

Heparan sulfate proteoglycans play a vital role in signaling of various growth factors in both Drosophila and vertebrates. In Drosophila, mutations in the tout velu (ttv) gene, a homolog of the mammalian EXT1 tumor suppressor gene, leads to abrogation of glycosaminoglycan (GAG) biosynthesis. This impairs distribution and signaling activities of various morphogens such as Hedgehog (Hh), Wingless (Wg), and Decapentaplegic (Dpp). Mutations in members of the exostosin (EXT) gene family lead to hereditary multiple exostosis in humans leading to bone outgrowths and tumors. In this study, we provide genetic and biochemical evidence that the human EXT1 (hEXT1) gene is conserved through species and can functionally complement the ttv mutation in Drosophila. The hEXT1 gene was able to rescue a ttv null mutant to adulthood and restore GAG biosynthesis.     

Prevalence,intensity and associated factor analysis of <Emphasis Type="Italic">Tropilaelaps</Emphasis><Emphasis Type="Italic">mercedesae</Emphasis> infesting <Emphasis Type="Italic">Apis</Emphasis><Emphasis Type="Italic">mellifera</Emphasis> in China

Tropilaelaps mercedesae is a serious ectoparasite of Apis mellifera in China. The aim of this study was to investigate the infestation rates and intensity of T. mercedesae in A. mellifera in China, and to explore the relative importance of climate, district, management practices and beekeeper characteristics that are assumed to be associated with the intensity of T. mercedesae. Of the 410 participating apiaries, 379 apiaries were included in analyses of seasonal infestation rates and 352 apiaries were included in multivariable regression analysis. The highest infestation rate (86.3%) of T. mercedesae was encountered in autumn, followed by summer (66.5%), spring (17.2%) and winter (14.8%). In autumn, 28.9% (93) of the infested apiaries were in the north (including the northeast and northwest of China), 71.1% (229) were in the central and south (including east, southeast and southwest China), and 306 apiaries (82.9%) were co-infested by both T. mercedesae and Varroa. Multivariable regression analysis showed that geographical location, season, royal jelly collection and Varroa infestation were the factors that influence the intensity of T. mercedesae. The influence of beekeeper’s education, time of beekeeping, operation size, and hive migration on the intensity of T. mercedesa was not statistically significant. This study provided information about the establishment of the linkage of the environment and the parasite and could lead to better timing and methods of control.     

Phenotypic Switching of <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis> and <Emphasis Type="Italic">Cryptococcus</Emphasis><Emphasis Type="Italic">gattii</Emphasis>

Microorganisms that live in fluctuating environments must constantly adapt their behavior to survive. The host constitutes an important microenvironment in opportunistic and primary fungal pathogens like Cryptococcus neoformans (C. neoformans) and Cryptococcus gattii (C. gattii). In clonal populations, adaptation may be achieved through the generation of diversity. For fungi phenotype switching constitutes a mechanism that allows them to change rapidly. Both C. neoformans and C. gattii undergo phenotypic switching, which allows them to be successful pathogens and cause persistent disease. Similar to other encapsulated microbes that exhibit phenotypic variation, phenotypic switching in Cryptococcus changes the polysaccharide capsule. Most importantly, in animal models phenotypic switching affects virulence and can change the outcome of infection. Virulence changes because C. neoformans and C. gattii switch variants elicit different inflammatory responses in the host. This altered host response can also affect the response to antifungal therapy and in some cases may even promote the selection of switch variants. This review highlights the similarity and differences between phenotypic switching in C. neoformans and C. gattii, the two dominant species that cause cryptococcosis in humans.