Electrophoretic karyotype and gene mapping of the vascular wilt fungus Verticillium dahliae

The karyotype profile of Verticillium dahliae was resolved by pulse-field gel electrophoresis. It revealed 6 chromosomal bands that corresponded to 7 chromosomes as shown by RFLP analysis using as probe the telomeric consensus sequence (AACCCT)(5). The number of chromosomes was further verified by the sensitivity of the hybridization signals to Bal31 digestion and the exclusion of interfering mitochondrial DNA signals. The corresponding sizes of the seven separated chromosomes were 6.7, 5.6, 4.1, 3.4, 3.1, 3.1 and 2.4Mb, raising the total genomic size of the fungus to approximately 28.4Mb. Twenty five homologous V. dahliae genes obtained either from randomly sequenced clones or PCR amplification were used as hybridization probes and were located onto the seven chromosomes.     

Effects of inducible overexpression of DNp73alpha on cancer cell growth and response to treatment in vitro and in vivo

The p73 gene has a complex regulation, which leads to the expression of different isoforms, often with opposite biological effects. We have generated in the human colocarcinoma cell line HCT116, expressing a wild-type p53, an inducible DNp73alpha expressing system. Two clones (HCT116/DN3 and HCT116/DN14), upon doxycycline addition, show a strong expression of DNp73alpha. In vitro the two DNp73alpha overexpressing clones grow at similar rate of the control transfected clone (HCT116/8a) and similarly respond to DNA damage. When injected in mice, HCT116/DN3, HCT116/DN14, and HCT116/8a cells grew similarly in the absence or presence of tetracycline. In HCT116/DN3 and HCT116/DN14 tumors, tetracycline induced a strong expression of DNp73alpha both as mRNA and protein. These results indicate that in this system the overexpression of the DNp73alpha does not induce a more aggressive phenotype and does not seem to be associated with a reduced response of the cells to treatment with anticancer agents.     

Coenzyme site-directed mutants of photosynthetic A4-GAPDH show selectively reduced NADPH-dependent catalysis, similar to regulatory AB-GAPDH inhibited by oxidized thioredoxin

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of higher plants uses both NADP(H) and NAD(H) as coenzyme and consists of one (GapA) or two types of subunits (GapA, GapB). AB-GAPDH is regulated in vivo through the action of thioredoxin and metabolites, showing higher kinetic preference for NADPH in the light than in darkness due to a specific effect on kcat(NADPH). Previous crystallographic studies on spinach chloroplast A4-GAPDH complexed with NADP or NAD showed that residues Thr33 and Ser188 are involved in NADP over NAD selectivity by interacting with the 2'-phosphate group of NADP. This suggested a possible involvement of these residues in the regulatory mechanism. Mutants of recombinant spinach GapA (A4-GAPDH) with Thr33 or Ser188 replaced by Ala (T33A, S188A and double mutant T33A/S188A) were produced, expressed in Escherichia coli, and compared to wild-type recombinant A4-GAPDH, in terms of crystal structures and kinetic properties. Affinity for NADPH was decreased significantly in all mutants, and kcat(NADPH) was lowered in mutants carrying the substitution of Ser188. NADH-dependent activity was unaffected. The decrease of kcat/Km of the NADPH-dependent reaction in Ser188 mutants resembles the behaviour of AB-GAPDH inhibited by oxidized thioredoxin, as confirmed by steady-state kinetic analysis of native enzyme. A significant expansion of size of the A4-tetramer was observed in the S188A mutant compared to wild-type A4. We conclude that in the absence of interactions between Ser188 and the 2'-phosphate group of NADP, the enzyme structure relaxes to a less compact conformation, which negatively affects the complex catalytic cycle of GADPH. A model based on this concept might be developed to explain the in vivo light-regulation of the GAPDH.     

Interrelationship between vitamins and cytokines in immunity

Cytokines are important proteins that modulate immunity and inflammation. Vitamins are also involved in immunity and inflammation. They are found to restore the ability of some cells to produce certain cytokines. Vitamin deficiency appears to affect the mechanism of immune cells, though the impact of reduced cytokine response in vitamin malnutrition is not clear. Vitamin D is involved in many medical conditions, such as infections and inflammation, and mediates innate immunity. Deficiency of vitamin D increases the risk of infectious and inflammatory diseases. In addition, this vitamin modulates Treg function and IL-10 production which is important for therapeutic treatment. Vitamin A increases inflammatory response and is involved in tissue damage; moreover, vitamin A is a key modulator of TGFbeta which can suppress several cytokines. Vitamin E, an anti-ageing compound, is associated with a defect of naive T cells and may inhibit some inflammatory compounds such as prostaglandin generation.     

Effects of AeDNV infection on Aedes aegypti lifespan and reproduction

The effects of Aedes Densovirus (AeDNV) infections on survival, fertility, fecundity and vertical transmission in Aedes aegypti (Diptera: Culicidae) were measured in laboratories in Kiev, Ukraine and Colorado, USA and incorporated into a predictive model of the effects of AeDNV on vector capacity. Adult lifespan and daily survival were reduced in AeDNV infected mosquitoes. This effect was dependent on the dose of the virus. Infected females had decreased fecundity. The oviposition rate was less in infected females and the hatch rate declined in eggs laid by infected females. The amounts of AeDNV in infected females and the infection rate of their offspring were measured with real-time PCR. The average filial transmission rate was 70% and larval infection rates from infected females varied between 42 and 62%. Vertically infected larvae, and individual eggs contained 1 × 10⁵ AeDNV genome equivalents (geq). Modeling the effects of AeDNV infection on Ae. aegypti populations suggested a large decrease in the numbers of eggs, larvae, pupae, and adults arising from infected mothers and suggested that AeDNV treatment of larvae could cause up to a 76% reduction of infectious mosquito days.     

Synthesis, characterization, crystal structures and in vitro antistaphylococcal activity of organotin(IV) derivatives with 5,7-disubstituted-1,2,4-triazolo[1,5-a]pyrimidine

New organotin(IV) complexes of 5,7-ditertbutyl-1,2,4-triazolo[1,5-a]pyrimidine (dbtp) and 5,7-diphenyl-1,2,4-triazolo[1,5-a]pyrimidine (dptp) with 1:1 and/or 1:2 stoichiometry were synthesized and investigated by X-ray diffraction, FT-IR and ¹¹⁹Sn Mössbauer in the solid state and by ¹H and ¹³C NMR spectroscopy, in solution. Moreover, the crystal and molecular structures of Et₂SnCl₂(dbtp)₂ and Ph₂SnCl₂(EtOH)₂(dptp)₂ are reported. The complexes contain hexacoordinated tin atoms: in Et₂SnCl₂(dbtp)₂ two 5,7-ditertbutyl-1,2,4-triazolo[1,5-a]pyrimidine molecules coordinate classically the tin atom through N(3) atom and the coordination around the tin atom shows a skew trapezoidal structure with axial ethyl groups. In Ph₂SnCl₂(EtOH)₂(dptp)₂ two ethanol molecules coordinate tin through the oxygen atom and the 5,7-diphenyl-1,2,4-triazolo[1,5-a]pyrimidine molecules are not directly bound to the metal center but strictly H-bonded, through N(3), to the OH group of the ethanol moieties; Ph₂SnCl₂(EtOH)₂(dptp)₂ has an all-trans structure and the C-Sn-C fragment is linear. On the basis of Mössbauer data, the 1:2 diorganotin(IV) complexes are advanced to have the same structure of Et₂SnCl₂(dbtp)₂, while Me₂SnCl₂(dptp)₂ to have a regular all-trans octahedral structure. A distorted cis-R₂ trigonal bipyramidal structure is assigned to 1:1 diorganotin(IV) complexes. The in vitro antibacterial activities of the synthesized complexes have been tested against a group of reference pathogen micro-organisms and some of them resulted active with MIC values of 5 μg/mL, most of all against staphylococcal strains, which shows their inhibitory effect.     

Hormone-induced 14-3-3γ adaptor protein regulates steroidogenic acute regulatory protein activity and steroid biosynthesis in MA-10 Leydig cells

Cholesterol is the sole precursor of steroid hormones in the body. The import of cholesterol to the inner mitochondrial membrane, the rate-limiting step in steroid biosynthesis, relies on the formation of a protein complex that assembles at the outer mitochondrial membrane called the transduceosome. The transduceosome contains several mitochondrial and cytosolic components, including the steroidogenic acute regulatory protein (STAR). Human chorionic gonadotropin (hCG) induces de novo synthesis of STAR, a process shown to parallel maximal steroid production. In the hCG-dependent steroidogenic MA-10 mouse Leydig cell line, the 14-3-3γ protein was identified in native mitochondrial complexes by mass spectrometry and immunoblotting, and its levels increased in response to hCG treatment. The 14-3-3 proteins bind and regulate the activity of many proteins, acting via target protein activation, modification and localization. In MA-10 cells, cAMP induces 14-3-3γ expression parallel to STAR expression. Silencing of 14-3-3γ expression potentiates hormone-induced steroidogenesis. Binding motifs of 14-3-3γ were identified in components of the transduceosome, including STAR. Immunoprecipitation studies demonstrate a hormone-dependent interaction between 14-3-3γ and STAR that coincides with reduced 14-3-3γ homodimerization. The binding site of 14-3-3γ on STAR was identified to be Ser-194 in the STAR-related sterol binding lipid transfer (START) domain, the site phosphorylated in response to hCG. Taken together, these results demonstrate that 14-3-3γ negatively regulates steroidogenesis by binding to Ser-194 of STAR, thus keeping STAR in an unfolded state, unable to induce maximal steroidogenesis. Over time 14-3-3γ homodimerizes and dissociates from STAR, allowing this protein to induce maximal mitochondrial steroid formation.     

Low 2-dimensional CD4 T cell receptor affinity for myelin sets in motion delayed response kinetics

T cells recognizing self-peptides that mediate autoimmune disease and those that are responsible for efficacious immunity against pathogens may differ in affinity for antigen due to central and peripheral tolerance mechanisms. Here we utilize prototypical self-reactive (myelin) and viral-specific (LCMV) T cells from T cell receptor (TCR) transgenic mice (2D2 and SMARTA, respectively) to explore affinity differences. The T cells responsive to virus possessed >10,000 fold higher 2D affinity as compared to the self-reactive T cells. Despite their dramatically lower affinity for their cognate ligand, 2D2 T cells respond with complete, albeit delayed, activation (proliferation and cytokine production). SMARTA activation occurs rapidly, achieving peak phosphorylation of p38 (1 minute), Erk (30 minutes), and Jun (3 hours) as well as CD69 and CD25 upregulation (3 and 6 hours, respectively), with a corresponding early initiation of proliferation. 2D2 stimulation with MOG results in altered signaling--no phospho-Erk or phospho-p38 accumulation, significantly delayed activation kinetics of Jun (12 hours), and delayed but sustained SHP-1 activity--as well as delayed CD69 and CD25 expression (12-24 hours), and slow initiation of proliferation. This delay was not intrinsic to the 2D2 T cells, as a more potent antigen with >100-fold increased 2D affinity restored rapid response kinetics in line with those identified for the viral antigen. Taken together, these data demonstrate that time can offset low TCR affinity to attain full activation and suggest a mechanism by which low affinity T cells participate in autoimmune disease.     

AFLP markers as a tool to reconstruct complex relationships: A case study in Rosa (Rosaceae)

The genus Rosa has a complex evolutionary history caused by several factors, often in conjunction: extensive hybridization, recent radiation, incomplete lineage sorting, and multiple events of polyploidy. We examined the applicability of AFLP markers for reconstructing (species) relationships in Rosa, using UPGMA clustering, Wagner parsimony, and Bayesian inference. All trees were well resolved, but many of the deeper branches were weakly supported. The cluster analysis showed that the rose cultivars can be separated into a European and an Oriental cluster, each being related to different wild species. The phylogenetic analyses showed that (1) two of the four subgenera (Hulthemia and Platyrhodon) do not deserve subgeneric status; (2) section Carolinae should be merged with sect. Cinnamomeae; (3) subsection Rubigineae is a monophyletic group within sect. Caninae, making sect. Caninae paraphyletic; and (4) there is little support for the distinction of the five other subsections within sect. Caninae. Comparison of the trees with morphological classifications and with previous molecular studies showed that all methods yielded reliable trees. Bayesian inference proved to be a useful alternative to parsimony analysis of AFLP data. Because of their genome-wide sampling, AFLPs are the markers of choice to reconstruct (species) relationships in evolutionary complex groups.     

Biodiversity and distribution of water mites (Acari, Hydrachnidia) in spring habitats

1. The distribution, species richness and ecology of spring‐dwelling water mites in Italy were investigated with the aim to better elucidate the role of spring habitats to sustain high levels of biodiversity and their contribution to freshwater biodiversity at a local and regional scale. 2. More than 300 springs in different geographic areas (Alps, Central and Southern Apennines, Sicily and Sardinia), were examined with a total of 163 water mite species recorded. 3. Species richness in each area ranged from 33 species on Sardinia to 77 on Sicily. The highest diversity was found in the Gran Sasso (Central Apennines). The proportion of crenobionts (species strictly bound to this type of habitat) exceeded 50% in almost all the areas investigated. 4. A diverse (up to 20 species per spring) and highly specialised mite fauna was observed in undisturbed rheocrenes and in natural springs of intermediate typology (rheohelocrenes and rheopsammocrenes). In springs subjected to human impacts (pasture, deforestation, alteration and transformation of spring sources) species richness declined and crenobionts were replaced by unspecialised crenoxenes. 5. The zoogeographic importance of spring habitats is confirmed by the presence of 18 endemic species and by members of genera with an interesting disjunct and relict distribution. 6. A comparison with other geographic areas suggests that springs contain a significant fraction of the total number of species found in freshwater habitats and may contribute almost one third of regional freshwater biodiversity. The presence of endemic crenobionts and rare taxa highlights the importance of these habitats in maintaining high levels of biodiversity as well as their contribution to a better understanding of biodiversity patterns in freshwaters.