Acetylcholine-activated currents in quail myotubes expressing viral oncogenes

Acetylcholine-activated currents were recorded in cultured myotubes arising from embryonic quail myoblasts transformed by the v-src and v-ras oncogenes. In src-myotubes, the whole cell inward current decayed more slowly than in non-transformed controls. In ras-myotubes, the current had a faster decay and smaller amplitude than in the controls. The single-channel conductance and mean open times recorded from cell-attached patches were similar in transformed and control cells. However, in ras-myotubes the frequency of channel openings strongly decreased with time.

It is concluded that oncogenic tyrosine-specific protein kinase (v-src product) and G-like p21 protein (v-ras product) can induce differential changes in the function of nicotinic ACh receptor, perhaps related to specific biochemical events elicited in the establishment of the transformed state.     

Effects of physiological self-crowding of DNA on shape and biological properties of DNA molecules with various levels of supercoiling

DNA in bacterial chromosomes and bacterial plasmids is supercoiled. DNA supercoiling is essential for DNA replication and gene regulation. However, the density of supercoiling in vivo is circa twice smaller than in deproteinized DNA molecules isolated from bacteria. What are then the specific advantages of reduced supercoiling density that is maintained in vivo? Using Brownian dynamics simulations and atomic force microscopy we show here that thanks to physiological DNA–DNA crowding DNA molecules with reduced supercoiling density are still sufficiently supercoiled to stimulate interaction between cis-regulatory elements. On the other hand, weak supercoiling permits DNA molecules to modulate their overall shape in response to physiological changes in DNA crowding. This plasticity of DNA shapes may have regulatory role and be important for the postreplicative spontaneous segregation of bacterial chromosomes.     

Reciprocal activating interaction between dendritic cells and pamidronate-stimulated gammadelta T cells: role of CD86 and inflammatory cytokines

We investigated the interactions between human monocyte-derived dendritic cells (DCs) and Ag-activated circulating TCR-gammadelta-expressing lymphocytes (Vdelta2). Coculture of immature DCs (iDCs) with peripheral blood Vdelta2 T cells activated with either pyrophosphomonoesters (isopentenyl pyrophosphate; IPP) or aminobiphosphonates (pamidronate; PAM) led to a significant up-modulation of CD86 and MHC class I molecules and to the acquisition of functional features typical of activated DCs. DC activation induced by both IPP- and PAM-stimulated gammadelta T cells was mostly mediated by TNF-alpha and IFN-gamma secreted by activated lymphocytes. However, the effect of PAM-activated gammadelta T cells, but not that of IPP-activated cells, required cell-to-cell contact. Reciprocally, activation of Vdelta2 T cells by PAM, but not by IPP, was dependent on cell contact with iDCs. In fact, when PAM-stimulated DC-gammadelta T cell cocultures were separated by a semipermeable membrane or treated with blocking anti-CD86 Abs, induction of CD25 and CD69 as well as IFN-gamma and TNF-alpha secretion by Vdelta2 cells were strongly reduced. These results demonstrate for the first time a bidirectional activating interaction between iDCs and PAM-stimulated gammadelta T lymphocytes, thus suggesting a potential adjuvant role of this early cross-talk in the therapeutic activity of aminobiphosphonate drugs.     

Chemical differences in volatiles between Melittis melissophyllum L. subsp. melissophyllum and subsp. albida (Guss) P. W. Ball (Lamiaceae) determined by solid-phase microextraction (SPME) coupled with GC/FID and GC/MS

Melittis melissophyllum (Lamiaceae) is a perennial herb, typical of woody places, occurring in Italy with two subspecies, i.e., melissophyllum and albida. So far, the classification of these two taxa was only based on morphology, i.e., the presence of glandular trichomes, the dimension of the leaves, and the number of teeth on each side as the main discriminant characters. To find marker compounds to chemically discriminate the subsp. melissophyllum with respect to the subsp. albida, a solid-phase microextraction SPME analysis coupled with GC/FID (=flame ionization detector) and GC/MS was carried out. SPME proved to be a chemotaxonomically useful technique that permitted a clearly differentiation of the two subspecies at headspace level. The subsp. melissophyllum was characterized by high amount of the mushroom alcohol oct-1-en-3-ol and the phenolic coumarin, whilst the subsp. albida exhibited a high content in monoterpenes and sesquiterpenes, α-pinene, sabinene, and (E)-caryophyllene being the major compounds. Multivariate chemometric techniques, such as cluster analysis (CA) and principal-component analysis (PCA), were used to support chemical data and characterize the population according to the taxonomy. In addition, the micromorphology and distribution of glandular trichomes of both subspecies were studied by scanning electron microscopy (SEM).     

Selective Strolls: Fixation and Extinction in Diploids Are Slower for Weakly Selected Mutations Than for Neutral Ones

In finite populations, an allele disappears or reaches fixation due to two main forces, selection and drift. Selection is generally thought to accelerate the process: a selected mutation will reach fixation faster than a neutral one, and a disadvantageous one will quickly disappear from the population. We show that even in simple diploid populations, this is often not true. Dominance and recessivity unexpectedly slow down the evolutionary process for weakly selected alleles. In particular, slightly advantageous dominant and mildly deleterious recessive mutations reach fixation slightly more slowly than neutral ones (at most 5%). This phenomenon determines genetic signatures opposite to those expected under strong selection, such as increased instead of decreased genetic diversity around the selected site. Furthermore, we characterize a new phenomenon: mildly deleterious recessive alleles, thought to represent a wide fraction of newly arising mutations, on average survive in a population slightly longer than neutral ones, before getting lost. Consequently, these mutations are on average slightly older than neutral ones, in contrast with previous expectations. Furthermore, they slightly increase the amount of weakly deleterious polymorphisms, as a consequence of the longer unconditional sojourn times compared to neutral mutations.     

Ultrastructure of the stylar canal cells ofCitrus limon (Rutaceae)

The ultrastructure of the canal cells and the canal filling substance ofCitrus limon have been studied. At maturity the canal cells are very rich in cytoplasm. Their inner tangential walls lining the canal are much thickened and formed by two layers: the outer corresponds to the original wall, the inner is formed by subsequent deposition of abundant materials of different origin. This thickening occurs at the same time as the filling of the stylar canal. Both events are paralleled by considerable dictyosomic activity, the formation of a large amount of rough endoplasmic reticulum, and the incorporation of small cytoplasmic masses into the cell wall, due to plasmalemma evaginations. — The material in the stylar canal has a heterogeneous ultrastructure aspect and consists of polysaccharides, proteins and lipids; it presumably provides nutrients for the growing pollen tubes.Research performed under CNR program Biology of Reproduction.     

The largest unassigned regions of the male- and female-transmitted mitochondrial DNAs in Musculista senhousia (Bivalvia Mytilidae)

Musculista senhousia is a marine mussel with doubly uniparental inheritance (DUI) of mitochondria. In this study we analyzed the largest unassigned region (LUR) of its female- and male-transmitted mitochondrial genomes, described their fine characteristics and searched for shared features. Our results suggest that both LURs contain the control region of their respective mitochondrial genomes. The female-transmitted control region is duplicated in tandem, with the two copies evolving in concert. This makes the F-mtDNA of M. senhousia the first Bivalve mitochondrial genome with this feature. We also compared M. senhousia control regions to that of other Mytilidae, and demonstrated that signals for basic mtDNA functions are retained over evolutionary times even among the fast-evolving mitochondrial genomes of DUI species. Finally, we discussed how similarities between female and male LURs may be explained in the context of DUI evolution and if the duplicated female control region might have influenced the DUI system in this species.     

Direct probing of RNA structure and RNA-protein interactions in purified HeLa cell's and yeast spliceosomal U4/U6.U5 tri-snRNP particles

The U4/U6.U5 tri-snRNP is a key component of spliceosomes. By using chemical reagents and RNases, we performed the first extensive experimental analysis of the structure and accessibility of U4 and U6 snRNAs in tri-snRNPs. These were purified from HeLa cell nuclear extract and Saccharomyces cerevisiae cellular extract. U5 accessibility was also investigated. For both species, data demonstrate the formation of the U4/U6 Y-shaped structure. In the human tri-snRNP and U4/U6 snRNP, U6 forms the long range interaction, that was previously proposed to be responsible for dissociation of the deproteinized U4/U6 duplex. In both yeast and human tri-snRNPs, U5 is more protected than U4 and U6, suggesting that the U5 snRNP-specific protein complex and other components of the tri-snRNP wrapped the 5' stem-loop of U5. Loop I of U5 is partially accessible, and chemical modifications of loop I were identical in yeast and human tri-snRNPs. This reflects a strong conservation of the interactions of proteins with the functional loop I. Only some parts of the U4/U6 Y-shaped motif (the 5' stem-loop of U4 and helix II) are protected. Due to difference of protein composition of yeast and human tri-snRNP, the U6 segment linking the 5' stem-loop to the Y-shaped structure and the U4 central single-stranded segment are more accessible in the yeast than in the human tri-snRNP, especially, the phylogenetically conserved ACAGAG sequence of U6. Data are discussed taking into account knowledge on RNA and protein components of yeast and human snRNPs and their involvement in splicesome assembly.