Multiple effects of paclitaxel are modulated by a high c-myc amplification level

Paclitaxel affects microtubule stability by binding to beta-tubulin, thus leading to cell accumulation in the G(2)/M phase, polyploidization, and apoptosis. Because both cell proliferation and apoptosis could be somehow regulated by the protooncogene c-myc, in this work we have investigated whether the c-myc amplification level could modulate the multiple effects of paclitaxel. To this aim, paclitaxel was administered to SW613-12A1 and -B3 human colon carcinoma cell lines (which are characterized by a high and low c-myc endogenous amplification level, respectively), and to the B3mycC5 cell line, with an enforced exogenous expression of c-myc copies. In this experimental system, we previously demonstrated that a high endogenous/exogenous level of amplification of c-myc enhances serum deprivation- and DNA damage-induced apoptosis. Accordingly, the present results indicate that a high c-myc amplification level potentiates paclitaxel cytotoxicity, confers a multinucleated phenotype, and promotes apoptosis to a great extent, thus suggesting that c-myc expression level is relevant in modulating the cellular responses to paclitaxel. We have recently shown in HeLa cells that the phosphorylated form of c-Myc accumulates in the nucleus, as distinct nucleolar and extranucleolar spots; here, we demonstrated that, after the treatment with paclitaxel, phosphorylated c-Myc undergoes redistribution, becoming diffused in the nucleoplasm.     

Microsporidiosis of Tachinaephagus zealandicus Ashmead (Hymenoptera: Encyrtidae)

An undescribed microsporidium was found infecting Tachinaephagus zealandicus, a gregarious parasitoid that attacks third instar larvae of muscoid flies. Spores were present in all body regions and in all stages of development. Infected adults contained an average of 3.75 x 10(5) spores, and the pathogen was vertically transmitted to progeny. Infected female adults were fed either rifampicin or albendazole mixed with honey to determine the effectiveness of these drugs in preventing vertical transmission. After eight days of feeding on rifampicin the parasitoids produced progeny of which only 37% were infected. In contrast, albendazole-treated and untreated females produced progeny that were 97% and 100% infected, respectively. Healthy and infected colonies were established and studies were conducted to determine the mechanisms of transmission. It was observed that the efficiency of vertical (maternal) transmission was 96.3%. Uninfected parasitoid immatures also became infected when they shared superparasitized hosts with infected immatures. The method of transmission within superparasitized hosts is not known.     

A new and distinctive male-sterile,female-fertile desynaptic mutant in soybean (Glycine max)

A spontaneous desynaptic mutation, affecting only microsporogenesis and causing pollen sterility, has been detected in BR97-12986H, a line of the official Brazilian soybean breeding program. In this male-sterile, female-fertile mutant, up to metaphase II, the meiotic behavior was similar to that described for the st series of synaptic mutants previously reported in soybean. Besides many univalents, few or total absence of bivalents were recorded in diakinesis. Bivalents presented one or two terminal chiasmata, while univalents retained the sister chromatid cohesion. Bivalents and most univalents congregated at the equatorial metaphase plate, although univalents frequently migrated to the poles prematurely. Laggards resulting from delay in chiasmata terminalization were also recorded. Distinctly different in their behavior from st series soybean mutants, telophase I-originated micronuclei of different sizes organized their own spindle in the second division. This behavior contributed towards an increase in genome fractionation. Several microspores and microcytes of different sizes were recorded at the end of meiosis. Pollen sterility was estimated at 91.2%. Segregation ratio for sterility in this line and its progenies reached 3:1. Allelism tests with st series of synaptic mutants are in progress. The importance of male-sterile, female-fertile mutations for soybean breeding programs is discussed.     

Regulation of the interleukin-1 receptor antagonist in THP-1 cells by ligands of the peroxisome proliferator-activated receptor gamma

Monocytes/macrophages (Mphi) play a pivotal role in the persistence of chronic inflammation and local tissue destruction in diseases such as rheumatoid arthritis and atherosclerosis. The production by Mphi of cytokines, chemokines, metalloproteinases and their inhibitors is an essential component in this process, which is tightly regulated by multiple factors. The peroxisome proliferator-activated receptors (PPARs) were shown to be involved in modulating inflammation. PPARgamma is activated by a wide variety of ligands such as fatty acids, the anti-diabetic thiazolidinediones (TZDs), and also by certain prostaglandins of which 15-deoxy-Delta(12,14)-PGJ2 (PGJ2). High concentrations of PPARgamma ligands were shown to have anti-inflammatory activities by inhibiting the secretion of interleukin-1 (IL-1), interleukin-6 (IL-6) and tumour necrosis factor alpha (TNFalpha) by stimulated monocytes.The aim of this study was to determine whether PGJ2 and TZDs would also exert an immunomodulatory action through the up-regulation of anti-inflammatory cytokines such as the IL-1 receptor antagonist (IL-1Ra). THP-1 monocytic cells were stimulated with PMA, thereby enhancing the secretion of IL-1, IL-6, TNFalpha, IL-1Ra and metalloproteinases. Addition of PGJ2 had an inhibitory effect on IL-1, IL-6 and TNFalpha secretion, while increasing IL-1Ra production. In contrast, the bona fide PPARgamma ligands (TZDs; rosiglitazone, pioglitazone and troglitazone) barely inhibited proinflammatory cytokines, but strongly enhanced the production of IL-1Ra from PMA-stimulated THP-1 cells. Unstimulated cells did not respond to TZDs in terms of IL-1Ra production, suggesting that in order to be effective, PPAR ligands depend on PMA signalling. Basal levels of PPARgamma are barely detectable in unstimulated THP-1 cells, while stimulation with PMA up-regulates its expression, suggesting that higher levels of PPARgamma expression are necessary for receptor ligand effects to occur. In conclusion, we demonstrate for the first time that TZDs may exert an anti-inflammatory activity by inducing the production of the IL-1Ra.     

Computer-based design of an HLA-haplotype and HIV-clade independent cytotoxic T-lymphocyte assay for monitoring HIV-specific immunity

BACKGROUND: Human immunodeficiency virus (HIV)- specific CD8-positive cytotoxic T-lymphocytes (CTL) play a key role in controlling HIV infection. Monitoring CTL response could be clinically relevant during structured therapy interruption (STI), HIV exposure, and vaccine trials. However, HLA patients' restriction and HIV variability limited the development of a CTL assay with broad specificity. MATERIALS AND METHODS: We designed an HLA-class I/HIV-1 clade independent assay for assessing HIV- specific CTL by using a computer-assisted selection ofthe CTL epitopes. Twenty-eight 15-mers were selected by peptide-binding motifs analysis using different databases (HIV-Immunology Database, SYFPEITHI, BIMAS). Altogether they putatively bind to more than 90% of HLA haplotypes in different populations, with an overall HIV-1 variability below 9%. The peptide pool was used as an antigen in an intracellular cytokine staining (ICS) assay for quantifying HIV-specific CTL response. RESULTS: The test can be performed using both fresh and cryopreserved peripheral blood mononuclear cells (PBMC), whereas GAG protein as antigen works only on fresh PBMC. A significantly higher CTL response with respect to HIV-negative controls was detected in all HIV-1 infected subjects of two groups of patients with different ethnicities (Caucasians and Africans) and coming from areas with different HIV-1 clade prevalences (clade B and A/G, respectively). In Caucasian patients, after month of STI, the number of HIV-1 specific CTL (2,896 +/- 2,780 IFN-gamma specific CD8 cells/ml) was significantly higher than that found at enrolment (2,125 +/- 4,426 IFN-gamma specific CD8 cells/ml, p< 0.05). CONCLUSIONS: These data indicate that this CTL assay is broadly specific and could represent a useful clinical tool for HIV immunodiagnostic independent of HLA-haplotype and HIV-clade variabilities.     

Glycerol-induced formation of the molten globule from acid-denatured cytochrome c: implication for hierarchical folding

At high concentration (98% or higher, v/v), glycerol induces collapse of acid-denatured cytochrome c into a compact state, the G_U state, showing a molten globule character. The G_U state possesses a nativelike -helix structure but a tertiary conformation less packed with respect to the native state. The spectroscopic properties of the G_U state closely resemble those of the molten globule stabilized by the organic solvent from the native protein (called the G_N state), indicating that glycerol can stabilize the molten globule of cytochrome c either from the native or the acid-denatured protein. The G_U and the G_N states show spectroscopic (and, thus, structural) properties and stabilities comparable to those of molten globules stabilized by different effectors, despite the fact that the mechanisms involved in the molten globule formation may significantly differ. This implies in cytochrome c a hierarchy for the rupture (native-to-molten globule) or the formation (unfolded-to-molten globule) of intramolecular interactions leading to the stabilization of the molten globule state of the protein, independently from the effector responsible for the structural transition, in accord with the sequential model proposed by Englander and collaborators.     

Urticalean rosids: circumscription, rosid ancestry, and phylogenetics based on rbcL, trnL-F, and ndhF sequences

To address the composition of the urticalean rosids, the relationships of the component families (maximally Cannabaceae, Cecropiaceae, Celtidaceae, Moraceae, Ulmaceae, and Urticaceae) and analyze evolution of morphological characters, we analyzed sequence variation for a large sampling of these families and various rosid outgroups using rbcL, trnL-F, and ndhF plastid regions. Urticalean rosids are derived out of a lineage including Barbeyaceae, Dirachmaceae, Elaeagnaceae, and Rhamnaceae, with Rosaceae less closely related; thus, they are imbedded within Rosales. Ulmaceae are the sister to all remaining families. Cannabaceae are derived out of a subclade of Celtidaceae; this expanded family should be called Cannabaceae. Cecropiaceae are derived within Urticaceae and are polyphyletic with Poikilospermum derived elsewhere within Urticaceae; this expanded family should be called Urticaceae. Monophyletic Moraceae are sister to this expanded Urticaceae. Support for these relationships comes from a number of morphological characters (floral sexuality, presence or absence of hypanthium, stamen type and dehiscence, pollen pore number, ovule position, and embryo alignment) and chromosome numbers. Most fruit types, in terms of ecological dispersal, are derived independently multiple times and are strongly correlated with habitat.     

PRC1 is a microtubule binding and bundling protein essential to maintain the mitotic spindle midzone

Midzone microtubules of mammalian cells play an essential role in the induction of cell cleavage, serving as a platform for a number of proteins that play a part in cytokinesis. We demonstrate that PRC1, a mitotic spindle-associated Cdk substrate that is essential to cell cleavage, is a microtubule binding and bundling protein both in vivo and in vitro. Overexpression of PRC1 extensively bundles interphase microtubules, but does not affect early mitotic spindle organization. PRC1 contains two Cdk phosphorylation motifs, and phosphorylation is possibly important to mitotic suppression of bundling, as a Cdk phosphorylation-null mutant causes extensive bundling of the prometaphase spindle. Complete suppression of PRC1 by siRNA causes failure of microtubule interdigitation between half spindles and the absence of a spindle midzone. Truncation mutants demonstrate that the NH2-terminal region of PRC1, rich in alpha-helical sequence, is important for localization to the cleavage furrow and to the center of the midbody, whereas the central region, with the highest sequence homology between species, is required for microtubule binding and bundling activity. We conclude that PRC1 is a microtubule-associated protein required to maintain the spindle midzone, and that distinct functions are associated with modular elements of the primary sequence.     

Genetic diversity of isolates of Glomus mosseae from different geographic areas detected by vegetative compatibility testing and biochemical and molecular analysis

We detected, for the first time, the occurrence of vegetative incompatibility between different isolates of the arbuscular mycorrhizal fungal species Glomus mosseae. Vegetative compatibility tests performed on germlings belonging to the same isolate showed that six geographically different isolates were capable of self-anastomosing, and that the percentage of hyphal contacts leading to fusions ranged from 60 to 85%. Successful anastomoses were characterized by complete fusion of hyphal walls, protoplasm continuity and occurrence of nuclei in the middle of hyphal bridges. No anastomoses could be detected between hyphae belonging to different isolates, which intersected without any reaction in 49 to 68% of contacts. Microscopic examinations detected hyphal incompatibility responses in diverse pairings, consisting of protoplasm retraction from the tips and septum formation in the approaching hyphae, even before physical contact with neighboring hyphae. Interestingly, many hyphal tips showed precontact tropism, suggesting that specific recognition signals may be involved during this stage. The intraspecific genetic diversity of G. mosseae revealed by vegetative compatibility tests was confirmed by total protein profiles and internal transcribed spacer-restriction fragment length polymorphism profiles, which evidenced a higher level of molecular diversity between the two European isolates IMA1 and BEG25 than between IMA1 and the two American isolates. Since arbuscular mycorrhizal fungi lack a tractable genetic system, vegetative compatibility tests may represent an easy assay for the detection of genetically different mycelia and an additional powerful tool for investigating the population structure and genetics of these obligate symbionts.     

Galactose-substituted alginate: preliminary characterization and study of gelling properties

Coupling of alginate with 1-amino-1-deoxygalactose in the presence of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide results in a substituted polymer containing galactose side linked via an amide bond. To clarify the degree and pattern of substitution, a (1)H NMR study on the anomeric region of modified alginate, polymannuronate, alginate enriched in guluronic acid (G-enriched alginate), and polyalternating MG, was carried out (G, alpha-l-guluronic acid; M, beta-d-mannuronic acid). From the resonance of the proton at position 1 of galactosylamine, it was possible to determine the amount of galactose linked to mannuronic and to guluronic residues, respectively. Furthermore, (1)H NMR spectroscopy revealed a higher reactivity of guluronic residues for low degrees of conversion. Modified alginates with 7% and 19% of substitution are both able to form stable beads in the presence of calcium ions. The effect of galactose substitution on the dimensions, swelling, and stability of the beads has been studied and the cytotoxicity of the modified polymer evaluated in preliminary biological tests.