Large impact of the apoplast on somatic embryogenesis in <Emphasis Type="Italic">Cyclamen persicum</Emphasis> offers possibilities for improved developmental control <Emphasis Type="Italic">in vitro</Emphasis>

Background  

Clonal propagation is highly desired especially for valuable horticultural crops. The method with the potentially highest multiplication rate is regeneration via somatic embryogenesis. However, this mode of propagation is often hampered by the occurrence of developmental aberrations and non-embryogenic callus. Therefore, the developmental process of somatic embryogenesis was analysed in the ornamental crop Cyclamen persicum by expression profiling, comparing different developmental stages of embryogenic cell cultures, zygotic vs. somatic embryos and embryogenic vs. non-embryogenic cell cultures.     

Purification and characterization of the α-glucosidase produced by thermophilic fungus <Emphasis Type="Italic">Thermoascus aurantiacus</Emphasis> CBMAI 756

An α-glucosidase enzyme produced by the fungus Thermoascus aurantiacus CBMAI 756 was purified by ultra filtration, ammonium sulphate precipitation, and chromatography using Q Sepharose, Sephacryl S-200, and Superose 12 columns. The apparent molecular mass of the enzyme was 83 kDa as determined in gel electrophoresis. Maximum activity was observed at pH 4.5 at 70°C. Enzyme showed stability stable in the pH range of 3.0–9.0 and lost 40% of its initial activity at the temperatures of 40, 50, and 60°C. In the presence of ions Na⁺, Ba²⁺, Co²⁺, Ni²⁺, Mg²⁺, Mn²⁺, Al³⁺, Zn²⁺, Ca²⁺ this enzyme maintained 90–105% of its maximum activity and was inhibited by Cr³⁺, Ag⁺, and Hg²⁺. The enzyme showed a transglycosylation property, by the release of oligosaccharides after 3 h of incubation with maltose, and specificity for short maltooligosaccharides and α-PNPG. The K_m measured for the α-glucosidase was 0.07 μM, with a V_max of 318.0 μmol/min/mg.     

Leishmania (Leishmania) infantum/chagasi: Histopathological aspects of the skin in naturally infected dogs in two endemic areas

In the New World, visceral leishmaniasis (VL), which is a progressive disease and frequently fatal, is caused by Leishmania (Leishmania) infantum/chagasi. It is endemic in many regions of Brazil and occasionally occurs in non-endemic regions when dogs from an endemic area are introduced. The aim of the present study is to compare different skin infection patterns of dogs from two leishmaniasis endemic areas. A histological analysis of dogs from Campo Grande, Mato Grosso do Sul state, a region where epidemic episodes are currently taking place, showed dermic inflammatory infiltrates, composed of numerous vacuolated parasitized macrophages, few lymphocytes, plasma cells and many degranulated mast cells. In the other region of the study, São Luís, Maranhão state, the skin of dogs presented a remarkable inflammatory reaction composed mainly of plasma cells, lymphocytes and very few parasites. We concluded that there is a difference in the skin lesion patterns of dogs with leishmaniasis that is directly related to the endemic area where the animals live.     

Age-related limitations of interleukin-6 in predicting early mortality in acute ST-elevation myocardial infarction

Interleukin-6 (IL-6) is an inflammatory cytokine whose levels increase significantly during myocardial infarction (MI).It has been hypothesised that the concentrations of IL-6 at admission may be useful in prognosticating long-term outcomes. It is unclear, however, whether IL-6 could improve the prognosis of early mortality in MI.We have compared serum IL-6 levels and analysed the disease course in 158 patients with ST-elevation MI (STEMI) who either survived (n?=?148) or died (n?=?10) within 30 days following the admission. Patients were treated in a single university centre with primary percutaneous coronary intervention (PCI).The non-survivors (6.3%) displayed most of typical risk factors for poor outcome. In addition they had significantly higher concentrations of IL-6 at hospital admission (median values 8.5 vs. 2.0 pg/ml; p?=?0.038). However, they were also significantly older than the survivors (median values 72 vs. 57 years; p?=?0.0001). IL-6 levels are known to increase with age and we could confirm a significant correlation between patients’ calendar age and circulating IL-6 (p?=?0.009). Regression analysis revealed that IL-6 concentrations were significantly affected by patients’ age but they did not independently relate to patients’ outcome.Such results indicate that circulating IL-6 at admission may be of limited value in predicting early mortality in STEMI. It is important to recognize that, because of the small group of patients who died (N?=?10), the results must be interpreted with caution. Therefore, we stress that these results should be viewed as preliminary and further validated in a larger set of patients.     

Comparative Study of 17-AAG and NVP-AUY922 in Pancreatic and Colorectal Cancer Cells: Are There Common Determinants of Sensitivity?

The use of heat shock protein 90 (Hsp90) inhibitors is an attractive antineoplastic therapy. We wanted to compare the effects of the benzoquinone 17-allylamino-17-demethoxygeldanamycin (17-AAG, tanespimycin) and the novel isoxazole resorcinol–based Hsp90 inhibitor NVP-AUY922 in a panel of pancreatic and colorectal carcinoma cell lines and in colorectal primary cultures derived from tumors excised to patients. PANC-1, CFPAC-1, and Caco-2 cells were intrinsically resistant to 17-AAG but sensitive to NVP-AUY922. Other cellular models were sensitive to both inhibitors. Human epidermal growth factor receptor receptors and their downstream signaling pathways were downregulated in susceptible cellular models, and concurrently, Hsp70 was induced. Intrinsic resistance to 17-AAG did not correlate with expression of ATP-binding cassette transporters involved in multidrug resistance. Some 17-AAG-resistant, NVP-AUY922–sensitive cell lines lacked NAD(P)H:quinone oxidoreductase 1 (NQO1) enzyme and activity. However, colorectal LoVo cells still responded to both drugs in spite of having undetectable levels and activity of NQO1. Pharmacological and biologic inhibition of NQO1 did not confer resistance to 17-AAG in sensitive cell lines. Therefore, even though 17-AAG sensitivity is related to NQO1 protein levels and enzymatic activity, the absence of NQO1 does not necessarily convey resistance to 17-AAG in these cellular models. Moreover, NVP-AUY922 does not require NQO1 for its action and is a more potent inhibitor than 17-AAG in these cells. More importantly, we show in this report that NVP-AUY922 potentiates the inhibitory effects of chemotherapeutic agents, such as gemcitabine or oxaliplatin, and other drugs that are currently being evaluated in clinical trials as antitumor agents.     

Sodium Hydrogen Exchangers Contribute to Arenavirus Cell Entry

Several arenaviruses, chiefly Lassa virus (LASV), cause hemorrhagic fever (HF) disease in humans and pose a great public health concern in the regions in which they are endemic. Moreover, evidence indicates that the worldwide-distributed prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen of clinical significance. The limited existing armamentarium to combat human-pathogenic arenaviruses underscores the importance of developing novel antiarenaviral drugs, a task that would be facilitated by the identification and characterization of virus-host cell factor interactions that contribute to the arenavirus life cycle. A genome-wide small interfering RNA (siRNA) screen identified sodium hydrogen exchanger 3 (NHE3) as required for efficient multiplication of LCMV in HeLa cells, but the mechanisms by which NHE activity contributed to the life cycle of LCMV remain unknown. Here we show that treatment with the NHE inhibitor 5-(N-ethyl-N-isopropyl) amiloride (EIPA) resulted in a robust inhibition of LCMV multiplication in both rodent (BHK-21) and human (A549) cells. EIPA-mediated inhibition was due not to interference with virus RNA replication, gene expression, or budding but rather to a blockade of virus cell entry. EIPA also inhibited cell entry mediated by the glycoproteins of the HF arenaviruses LASV and Junin virus (JUNV). Pharmacological and genetic studies revealed that cell entry of LCMV in A549 cells depended on actin remodeling and Pak1, suggesting a macropinocytosis-like cell entry pathway. Finally, zoniporide, an NHE inhibitor being explored as a therapeutic agent to treat myocardial infarction, inhibited LCMV propagation in culture cells. Our findings indicate that targeting NHEs could be a novel strategy to combat human-pathogenic arenaviruses.     

Ultrastructure of the ovarian follicles in the placentotrophic Andean lizard of the genus Mabuya (Squamata: Scincidae)

We studied the ultrastructural organization of the ovarian follicles in a placentotrophic Andean lizard of the genus Mabuya. The oocyte of the primary follicle is surrounded by a single layer of follicle cells. During the previtellogenic stages, these cells become stratified and differentiated in three cell types: small, intermediate, and large globoid, non pyriform cells. Fluid‐filled spaces arise among follicular cells in late previtellogenic follicles and provide evidence of cell lysis. In vitellogenic follicles, the follicular cells constitute a monolayered granulosa with large lacunar spaces; the content of their cytoplasm is released to the perivitelline space where the zona pellucida is formed. The oolemma of younger oocytes presents incipient short projections; as the oocyte grows, these projections become organized in a microvillar surface. During vitellogenesis, cannaliculi develop from the base of the microvilli and internalize materials by endocytosis. In the juxtanuclear ooplasm of early previtellogenic follicles, the Balbiani's vitelline body is found as an aggregate of organelles and lipid droplets; this complex of organelles disperses in the ooplasm during oocyte growth. In late previtellogenesis, membranous organelles are especially abundant in the peripheral ooplasm, whereas abundant vesicles and granular material occur in the medullar ooplasm. The ooplasm of vitellogenic follicles shows a peripheral band constituted by abundant membranous organelles and numerous vesicular bodies, some of them with a small lipoprotein core. No organized yolk platelets, like in lecithotrophic reptiles, were observed. Toward the medullary ooplasm, electron‐lucent vesicles become larger in size containing remains of cytoplasmic material in dissolution. The results of this study demonstrate structural similarities between the follicles of this species and other Squamata; however, the ooplasm of the mature oocyte of Mabuya is morphologically similar to the ooplasm of mature oocytes of marsupials, suggesting an interesting evolutionary convergence related to the evolution of placentotrophy and of microlecithal eggs. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.     

Regulation of ploidy and senescence by the AMPK‐related kinase NUAK1

Senescence is an irreversible cell‐cycle arrest that is elicited by a wide range of factors, including replicative exhaustion. Emerging evidences suggest that cellular senescence contributes to ageing and acts as a tumour suppressor mechanism. To identify novel genes regulating senescence, we performed a loss‐of‐function screen on normal human diploid fibroblasts. We show that downregulation of the AMPK‐related protein kinase 5 (ARK5 or NUAK1) results in extension of the cellular replicative lifespan. Interestingly, the levels of NUAK1 are upregulated during senescence whereas its ectopic expression triggers a premature senescence. Cells that constitutively express NUAK1 suffer gross aneuploidies and show diminished expression of the genomic stability regulator LATS1, whereas depletion of NUAK1 with shRNA exerts opposite effects. Interestingly, a dominant‐negative form of LATS1 phenocopies NUAK1 effects. Moreover, we show that NUAK1 phosphorylates LATS1 at S464 and this has a role in controlling its stability. In summary, our work highlights a novel role for NUAK1 in the control of cellular senescence and cellular ploidy.     

Weathering-Associated Bacteria from the Damma Glacier Forefield: Physiological Capabilities and Impact on Granite Dissolution

Several bacterial strains isolated from granitic rock material in front of the Damma glacier (Central Swiss Alps) were shown (i) to grow in the presence of granite powder and a glucose-NH₄Cl minimal medium without additional macro- or micronutrients and (ii) to produce weathering-associated agents. In particular, four bacterial isolates (one isolate each of Arthrobacter sp., Janthinobacterium sp., Leifsonia sp., and Polaromonas sp.) were weathering associated. In comparison to what was observed in abiotic experiments, the presence of these strains caused a significant increase of granite dissolution (as measured by the release of Fe, Ca, K, Mg, and Mn). These most promising weathering-associated bacterial species exhibited four main features rendering them more efficient in mineral dissolution than the other investigated isolates: (i) a major part of their bacterial cells was attached to the granite surfaces and not suspended in solution, (ii) they secreted the largest amounts of oxalic acid, (iii) they lowered the pH of the solution, and (iv) they formed significant amounts of HCN. As far as we know, this is the first report showing that the combined action of oxalic acid and HCN appears to be associated with enhanced elemental release from granite, in particular of Fe. This suggests that extensive microbial colonization of the granite surfaces could play a crucial role in the initial soil formation in previously glaciated mountain areas.Glaciers in alpine regions are highly sensitive to changes in climatic conditions (29). Increasing global atmospheric temperatures over the last decades have resulted in the recession of alpine glaciers (18). Forefields of temperate alpine glaciers provide unique opportunities to study initial soil formation as well as microbial and plant succession along the chronosequences (12, 26, 34, 36). The forefields close to the glacier terminus are initially vegetation free and consist mainly of rock material with high fractions of silt-sized grains with low C and N content and small amounts of available nutrients (14). Mineral weathering is a key process in the formation of soils (1, 26), and the crucial importance of microbially promoted mineral weathering for nutrient acquisition is increasingly recognized (2, 4, 39, 46). Recently exposed rock surfaces can be considered primary ecosystems where only a few microbes that are adapted due to their mineral-weathering abilities can grow (17). Some cations of rock-forming minerals are essential for proper cell functions. However, our understanding of geochemically significant microbes in forefields of temperate alpine glacier is still very limited but is crucial for increasing our knowledge of nutrient mobilization and the buildup of organic matter that is essential for the development of macroorganisms.The area of the Damma glacier in Central Switzerland is characterized by a relatively homogenous granitic rock basement and is used as field site of the interdisciplinary research project “Biosphere-Geosphere interactions: Linking climate change, weathering, soil formation and ecosystem evolution (BigLink)” (5). In the frame of this research project, we studied the functional roles of granite-colonizing microbes as biotic weathering agents in previously glaciated areas. So far, relatively little is known about microbe-granite interactions, especially regarding the release of trace elements. Several studies have examined the dissolution of specific granite-forming minerals in the presence of actively metabolizing bacteria or compounds that simulate metabolic activity (24, 30, 31, 37, 38, 44). There is a general agreement that microbially produced organic acids, siderophores, and extracellular polysaccharides can all promote dissolution of minerals. Previous dissolution experiments have mainly been performed with (i) commercially obtained minerals (23, 45), (ii) model microorganisms that were commercially obtained from culture collections (3, 35, 45), or (iii) laboratory strains, such as those of Bacillus subtilis (23) and Burkholderia fungorum (47). Most studies have focused on individual mineral specimens rather than on the mixture of minerals that are present in granite rock (47). Few studies observed mineral weathering of collected rock and bacteria isolated from volcanic areas covered with vegetation (30, 31). Moreover, there are no studies on microbial weathering for such immediately deglaciated environments combining functional and taxonomic investigations, probably due to the difficulties in obtaining heterotrophic bacterial isolates from granitic glacier forefields. In spite of this, a comprehensive culture collection containing approximately 500 bacterial strains, which were isolated from the glacier tongue of the Damma glacier, was established. Full-length 16S rRNA gene sequences of 120 isolates revealed that many isolates obtained from oligotrophic media were closely related to readily cultivable heterotrophic bacteria (e.g., Arthrobacter sp., Collimonas sp., Paenibacillus sp., and Pseudomonas sp.). These bacteria have been found to enhance mineral dissolution (39).Our aim was to characterize the impact of microorganisms on granite weathering. We performed laboratory dissolution experiments with sterile crushed granite rock material, 12 bacterial strains, and 1 algal strain. To investigate the potential weathering abilities of these isolates, granite dissolution experiments were performed abiotically with model agents, such as HCl for proton-promoted weathering or oxalate and citrate and KCN for ligand-promoted weathering.