The CR3 motif of Rrp44p is important for interaction with the core exosome and exosome function

The 10-subunit RNA exosome is involved in a large number of diverse RNA processing and degradation events in eukaryotes. These reactions are carried out by the single catalytic subunit, Rrp44p/Dis3p, which is composed of three parts that are conserved throughout eukaryotes. The exosome is named for the 3′ to 5′ exoribonuclease activity provided by a large C-terminal region of the Rrp44p subunit that resembles other exoribonucleases. Rrp44p also contains an endoribonuclease domain. Finally, the very N-terminus of Rrp44p contains three Cys residues (CR3 motif) that are conserved in many eukaryotes but have no known function. These three conserved Cys residues cluster with a previously unrecognized conserved His residue in what resembles a metal-ion-binding site. Genetic and biochemical data show that this CR3 motif affects both endo- and exonuclease activity in vivo and both the nuclear and cytoplasmic exosome, as well as the ability of Rrp44p to associate with the other exosome subunits. These data provide the first direct evidence that the exosome-Rrp44p interaction is functionally important and also provides a molecular explanation for the functional defects when the conserved Cys residues are mutated.     

Liver Accumulation of Plasmodium chabaudi-Infected Red Blood Cells and Modulation of Regulatory T Cell and Dendritic Cell Responses

It is postulated that accumulation of malaria-infected Red Blood Cells (iRBCs) in the liver could be a parasitic escape mechanism against full destruction by the host immune system. Therefore, we evaluated the in vivo mechanism of this accumulation and its potential immunological consequences. A massive liver accumulation of P. c. chabaudi AS-iRBCs (Pc-iRBCs) was observed by intravital microscopy along with an over expression of ICAM-1 on day 7 of the infection, as measured by qRT-PCR. Phenotypic changes were also observed in regulatory T cells (Tregs) and dendritic cells (DCs) that were isolated from infected livers, which indicate a functional role for Tregs in the regulation of the liver inflammatory immune response. In fact, the suppressive function of liver-Tregs was in vitro tested, which demonstrated the capacity of these cells to suppress naive T cell activation to the same extent as that observed for spleen-Tregs. On the other hand, it is already known that CD4+ T cells isolated from spleens of protozoan parasite-infected mice are refractory to proliferate in vivo. In our experiments, we observed a similar lack of in vitro proliferative capacity in liver CD4+ T cells that were isolated on day 7 of infection. It is also known that nitric oxide and IL-10 are partially involved in acute phase immunosuppression; we found high expression levels of IL-10 and iNOS mRNA in day 7-infected livers, which indicates a possible role for these molecules in the observed immune suppression. Taken together, these results indicate that malaria parasite accumulation within the liver could be an escape mechanism to avoid sterile immunity sponsored by a tolerogenic environment.     

Distribution of Adult Male and Female Baccharis concinna (Asteraceae) in the Rupestrian Fields of Serra Do Cipó, Brazil

Abstract: This study focuses on the sex ratio and spatial distribution of males and females in three populations of the endemic and restricted tropical dioecious shrub, Baccharis concinna (Asteraceae) in the mountainous region of Serra do Cipó, southeastern Brazil. The proportion of female plants in the population at lower elevation (1000 m a.s.l.) was significantly greater than of male plants. At this elevation of P/N and Ca/Al ratios in the soil were also greater indicating better nutritional status of the soils. The concentration of aluminium increased significantly with the elevation ( p < 0.001), perhaps rendering soils less conducive to female plants at higher elevations. Female plants are possibly adversely affected to a greater extent by soil quality than male plants. The spatial distribution of the populations within habitat was tested by the K(t) function, where the neighbourhood of a given individual was defined by a circle with a radius (t) up to 3 m. Despite the strong tendency for aggregation, the distribution of the sexes within habitats was random and the hypothesis was not supported. The independent distribution of the sexes within habitats may be explained by nutrient homogeneity of the soils, as well as by an absence of antagonism between the sexes. Nevertheless, we found a trend for males and females to be aggregated according to their gender.     

Relative Dominance Affects Use of Scent-Marked Areas in Male Snow Voles Chionomys nivalis

We conducted a laboratory experiment to examine whether dominance status affects the use of locations occupied (i.e. scent-marked) by same-sex conspecifics among wild-caught snow vole males ( Chionomys nivalis ). Given that the costs of invading scent-marked areas should partially depend on the intruder's competitive ability, we hypothesised that, once a dominance relationship has been established with the owner of marks, the use of these areas by males would vary differently between dominant and subordinate individuals. Before any previous experience with the owner, scented substrates and nests were highly attractive to all males, indicating a general preference for recently occupied areas. However, after relative social status was established through direct interaction the subsequent response of males was altered differently, subordinate individuals reducing the use of marked areas to a much greater extent than dominants. Competitive relationships between male C. nivalis were found to be influenced by differences in body weight, larger males tending to display a more dominant pattern of behaviour. Our results reveal that male C. nivalis may require some direct experience with potential opponents to modulate their response towards occupied locations. Additionally, we suggest that the ability of males to conditionally respond to social signals from particular competing conspecifics might be used to lower the costs of prospective agonistic interactions.     

Calcium-Dependent Release of Tyrosine in Brain Elicited by Stimulation of Neuropeptide Receptors

We sought to establish whether the endogenous opiate-receptor agonist Met-enkephalin (m-ENK) selectively modulates the release of endogenous tyrosine (Tyr) from brain slices prepared from the corpus striatum (CS). Amino acids (AAs) released from slices of CS and, for comparison, cerebral cortex (Cx) were measured by HPLC. Incubation of slices with m-ENK (1-10 microM) increased the basal release of Tyr (up to 293% of control) from CS, but not Cx, whereas other nonneurotransmitter AAs, phenylalanine (Phe) and valine (Val), were unchanged. The release of the putative neurotransmitter AAs glutamate (Glu), taurine (Tau), and glycine (Gly) were similarly increased by 50-150% with m-ENK in slices of CS, but not Cx. The enhanced release of AAs by m-ENK was prevented by removal of extracellular Ca2+ or by preincubation with the opiate receptor antagonist naloxone. Neuronal depolarization by potassium (5-55 mM) in the presence of Ca2+ did not affect the release of Tyr, whereas release of neurotransmitter AAs such as gamma-aminobutyric acid (GABA) were markedly increased. The increase in basal Tyr release by m-ENK was not the result of a decreased uptake of Tyr. Relative to slices, the basal release of Tyr, Phe, and Val from a synaptosomal (P2) preparation of CS was small (8-51%) compared to that of GABA, Gly, Glu, and Tau (49-123%). Nonetheless, m-ENK (10 microM) markedly increased the release of Tyr (to 833%), but not Glu, Gly, and Tau from the P2 fraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Homologous plasmid recombination is elevated in immortally transformed cells.

The levels of intramolecular plasmid recombination, following transfection of a plasmid substrate for homologous recombination into normal and immortally transformed cells, have been examined by two independent assays. In the first assay, recovered plasmid was tested for DNA rearrangements which regenerate a functional neomycin resistance gene from two overlapping fragments. Following transformation of bacteria, frequencies of recombinationlike events were determined from the ratio of neomycin-resistant (recombinant) colonies to ampicillin-resistant colonies (indicating total plasmid recovery). Such events, yielding predominantly deletions between the directly repeated sequences, were substantially more frequent in five immortal cell lines than in any of three normal diploid cell strains tested. Effects of plasmid replication or interaction with T antigen and of bacterially mediated rejoining of linear molecules generated in mammalian cells were excluded by appropriate controls. The second assay used limited coamplification of a control segment of plasmid DNA, and of the predicted recombinant DNA region, primed by two sets of flanking oligonucleotides. Each amplified band was quantitated by reference to a near-linear standard curve generated concurrently, and recombination frequencies were determined from the ratio of recombinant/control DNA regions. The results confirmed that recombinant DNA structures were generated within human cells at direct repeats in the transfected plasmid and were markedly more abundant in an immortal cell line than in the diploid normal cells from which that line was derived.     

The adsorption of Fusarium flocciferum spores on celite particles and their use in the degradation of phenol

Summary Spores of Fusarium flocciferum were inserted in porous celite beads. The effects of bead size, adsorption time course, washing cycle and spore concentration on spore loading were investigated. Cell loadings up to 50% (dry weight/beads) were obtained. The degradation of phenol using adsorbed cells was studied in batch experiments. The immobilized cell system was shown to efficiently degrade high concentrations of the substrate (up to 2.0 g/l) and to remain active for more than 2 motths. The oxygen uptake rate of free and immobilized cells was determined at various concentrations of phenol. The kinetic constants K _s=85 mg/l, K _i=345 mg/l and SMI=170 mg/l were estimated from the experimental data by linearization of the Haldane function for the free cells. The uptake rates exhibited by the confined cells were lower (30%) than those obtained for free cells and no significant differences were found for phenol concentrations between 150 and 1200 mg/l.     

Spontaneous release of endogenous aspartate and glutamate from rat striatal slices is increased following destruction of local neurons by ibotenic acid

We sought to determine in rat striatum whether the release of neurotransmitter amino acids aspartate (Asp), glutamate (Glu) and gamma-aminobutyric acid (GABA) were affected by local neurons. To do so, unilateral microinjections of ibotenic acid, an excitotoxin that destroys local neurons without affecting fibers of passage, were made into the striatum. Release of endogenous amino acids from lesioned and intact striatal slices were measured by HPLC one week later. The effectiveness and specificity of the lesion were confirmed by measuring the enzyme activity associated with extrinsic dopamine neurons (tyrosine hydroxylase; 111±14%), intrinsic GABA neurons (glutamic acid decarboxylase; 19±7%) and intrinsic acetylcholine neurons (choline acetyltransferase; 37±10%). Destruction of local striatal neurons markedly attenuated the release of GABA (41±12% of control) elicited by depolarization with K⁺ (35 mM), but did not significantly reduce the K⁺-evoked release of Asp (80±17%) and Glu (92±8%). However, spontaneous release of Asp and Glu was significantly greater than that observed in unlesioned tissue (159±18% and 209±27%, respectively), while the spontaneous release of GABA was not significantly reduced (75±43%). Although release of the neurotransmitter amino acids Asp, Glu and GABA were affected by the lesion, the release of the non-neurotransmitter amino acid tyrosine was unaffected. These data are consistent with the hypotheses that: 1) the predominant source of releasable stores of endogenous Asp and Glu in the striatum arises from extinsic neurons, and 2) that the spontaneous release of Asp and Glu from axon terminals in the striatum may be regulated, at least in part, by local inhibitory neurons.