Epstein-Barr virus mRNA export factor EB2 is essential for production of infectious virus

The splicing machinery which positions a protein export complex near the exon-exon junction mediates nuclear export of mRNAs generated from intron-containing genes. Many Epstein-Barr virus (EBV) early and late genes are intronless, and an alternative pathway, independent of splicing, must export the corresponding mRNAs. Since the EBV EB2 protein induces the cytoplasmic accumulation of intronless mRNA, it is tempting to speculate that EB2 is a viral adapter involved in the export of intronless viral mRNA. If this is true, then the EB2 protein is essential for the production of EBV infectious virions. To test this hypothesis, we generated an EBV mutant in which the BMLF1 gene, encoding the EB2 protein, has been deleted (EBV(BMLF1-KO)). Our studies show that EB2 is necessary for the production of infectious EBV and that its function cannot be transcomplemented by a cellular factor. In the EBV(BMLF1-KO) 293 cells, oriLyt-dependent DNA replication was greatly enhanced by EB2. Accordingly, EB2 induced the cytoplasmic accumulation of a subset of EBV early mRNAs coding for essential proteins implicated in EBV DNA replication during the productive cycle. Two herpesvirus homologs of the EB2 protein, the herpes simplex virus type 1 protein ICP27 and, the human cytomegalovirus protein UL69, only partly rescued the phenotype of the EBV(BMLF1-KO) mutant, indicating that some EB2 functions in virus production cannot be transcomplemented by ICP27 and UL69.     

The three polymorphic chains of human Hb-F studied in normal Italian newborns

Both the known polymorphisms of human Hb-F have been investigated here. Two similar groups of full-term newborns have been considered: pure F-globin has been studied by suitable fingerprinting techniques in the first group, while isoelectrofocusing techniques have been employed in the second. The results clearly indicate that G/A gamma polymorphism can be correctly determined with both the procedures, while the T/I gamma polymorphism cannot, because IEF is not sensitive enough towards low quantities of A gamma T chains. Moreover, in the first group of newborns, the A gamma T gene frequency (P) is 0.23, while the total G/A gamma ratio of both groups is about 7/3, the two data being in full agreement with those of the literature. Furthermore, additional reports show that the main functional parameters of the whole neonatal blood are not significantly different either in the presence or in the absence of A gamma T chains.     

Proteomic evaluation of sheep serum proteins

Background

Mammary tumours frequently develop in female domestic cats being highly malignant in a large percentage of cases. Chemokines regulate many physiological and pathological processes including organogenesis, chemotaxis of inflammatory cells, as well as tumour progression and metastasization. In particular, the chemokine/receptor pair SDF-1/CXCR4 has been involved in the regulation of metastatic potential of neoplastic cells, including breast cancer. The aim of this study was the immunohistochemical defininition of the expression profile of CXCR4 in primary and metastatic feline mammary carcinomas and the evaluation of the role of SDF-1 in feline mammary tumour cell proliferation.

Results

A total of 45 mammary surgical samples, including 33 primary tumours (31 carcinomas and 2 adenomas), 6 metastases, and 4 normal mammary tissues were anlyzed. Tumor samples were collected from a total number of 26 animals, as in some cases concurrent occurrence of neoplasm in more than one mammary gland was observed. Tissues were processed for standard histological examination, and all lesions were classified according to the World Health Organization criteria. CXCR4 expression in neoplastic cells was evaluated by immunohistochemistry. The level of CXCR4 immunoreactivity was semi-quantitatively estimated as CXCR4 score evaluating both the number of positive cells and the intensity of staining. Six primary, fibroblast-free primary cultures were obtained from fresh feline mammary carcinomas and characterized by immunofluorescence for CXCR4 and malignant mammary cell marker expression. SDF-1-dependent in vitro proliferative effects were also assayed. CXCR4 expression was observed in 29 out of 31 malignant tissues with a higher CXCR4 score observed in 4 out of 6 metastatic lesions than in the respective primary tumours. In 2 benign lesions analyzed, only the single basaloid adenoma showed a mild positive immunostaining against CXCR4. Normal tissue did not show CXCR4 immunoreactivity. CXCR4 score was statistically significantly associated with the histological features of the samples, showing an increase accordingly with the degree of neoplastic transformation (from normal tissue to metastatic lesions). Finally, in the primary cultures obtained from 6 primary feline mammary carcinomas CXCR4 expression was detected in all cells and its activation by SDF-1 in vitro treatment caused a significant increase in the proliferation rate in 5 out of 6 tumours.

Conclusions

These results indicate that malignant feline mammary tumours commonly express CXCR4, with a higher level in malignant tumours, and, in most of the cases analysed, metastatic cells display stronger immunoreactivity for CXCR4 than the corresponding primary tumours. Moreover, CXCR4 activation in primary cultures of feline mammary carcinomas causes increase in the proliferative rate. Thus, SDF-1/CXCR4 system seems to play a tumorigenic in feline mammary gland malignancy and in vitro cultures from these tumour samples may represent an experimental model to investigate the biological and pharmacological role of this chemokinergic axis.     

Suppression of arbuscular mycorrhizal colonization and nodulation in split-root systems of alfalfa after pre-inoculation and treatment with Nod factors

Roots of legumes establish symbiosis with arbuscular mycorrhizal fungi (AMF) and nodule-inducing rhizobia. The existing nodules systemically suppress subsequent nodule formation in other parts of the root, a phenomenon termed autoregulation. Similarly, mycorrhizal roots reduce further AMF colonization on other parts of the root system. In this work, split- root systems of alfalfa (Medicago sativa) were used to study the autoregulation of symbiosis with Sinorhizobium meliloti and the mycorrhizal fungus Glomus mosseae. It is shown that nodulation systemically influences AMF root colonization and vice versa. Nodules on one half of the split-root system suppressed subsequent AMF colonization on the other half. Conversely, root systems pre-colonized on one side by AMF exhibited reduced nodule formation on the other side. An inhibition effect was also observed with Nod factors (lipo-chito-oligosaccharides). NodSm-IV(C16:2, S) purified from S. meliloti systemically suppressed both nodule formation and AMF colonization. The application of Nod factors, however, did not influence the allocation of (14)C within the split-root system, excluding competition for carbohydrates as the regulatory mechanism. These results indicate a systemic regulatory mechanism in the rhizobial and the arbuscular mycorrhizal association, which is similar in both symbioses.     

Settler agriculture and the dynamics of resource allocation in frontier environments

This article attempts to conceptualize the dynamics of resource allocation by colonist farmers under the unique conditions of land abundance and labor scarcity which characterize frontier environments, such as the smallholder agricultural settlement areas in the Amazon basin. In contrast, most previous theoretical literature on household agricultural decision making and land-use change in rural areas considers conditions of high population density and land scarcity, and is not, therefore, adequate for understanding critical land-use changes which may be occurring in frontier regions. This article first discusses the appropriateness and inadequacies of the analytical frameworks commonly used to explain the expansion of settler agriculture into remote forest regions and the unsustainable land-use practices observed in these areas. This review serves as the basis for characterizing resource allocation under the particular conditions of frontier environments. A conceptual advance in the analysis is its consideration of the way institutional/policy factors and farm-level characteristics can interact to produce land-use outcomes. This knowledge is essential to understand not only the social and economic factors affecting present land use and choice of technology, but also those factors influencing farmers' demand for more optimal systems of land use which are consistent with varying agro-ecological potentials, demographic situations, and their own management capacity.     

Population structure and mycelial phenotypic variability of the ectomycorrhizal basidiomycete Laccaria bicolor (Maire) Orton

Mating type allele distribution and phenotypic variability were investigated in field populations of Laccaria bicolor. Sporophores associated with Norway spruce (Picea abies), colonized by natural sources of inoculum and growing in a seed orchard, were sampled to obtain dikaryotic strains and assay their phenotypic variability for traits important to the symbiosis. Basid-iospores were also collected for mating type analysis of different mycelia. Four sporophore mating types were identified containing seven A and five B factors. Out-breeding efficiency was estimated at 73.8% and the population was slightly inbred. Crosses with previously characterized L. bicolor strains from two nearby populations identified in total six sporophore mating types and ten A and nine B factors, for an estimated outbreeding efficiency (85.7%) similar to previous studies of more spatially disparate Laccaria spp. populations. Dikaryotic strains were tested for mycelial growth rate, as a measure of their competitive ability, on agar media containing a soluble (NaH₂PO₄), or an insoluble (CaHPO₄) phosphate source. Their ability to solubilize the latter was also tested to assess their relative capacity to access insoluble, inorganic phosphate. In most cases, significant variation was detected among strains from the same site for all variables. On three sites (VC4, VC5 and VC7), each determined previously to possess a uniform mycelial genotype, phenotypic variability was likely due to epigenetic variation among different strains of the same genotype. Possible evidence for dikaryon-monokaryon crosses was observed in vivo on one sample site (VC2) where adjacent mycelia shared two mating factors. The phenotypic variability of different mycelial genotypes reflected their genetic variability observed as mating type allele diversity and underlined the importance of basidiospore dispersal in introducing new genotypes into the population. The reproductive strategies of L. bicolor are discussed and compared to those of other basidiomycete species.     

The politics of numbers: Quebec’s historical struggle with ethnic and linguistic categories*

The production of indicators is intimately linked to the political context and therefore meets a social demand based on historical issues. Quebec is no exception. The use of official ethnic and linguistic categories has evolved from a purely ethnic perspective based on ethnic ancestry aimed at assessing the demographic balance between French and English (1860–1960) to linguistic categories with a strong ethnic content defined by mother tongue and language spoken at home (1960–1990). In the more recent period (since the 1990s), the official linguistic policy in an increasing diversified population aimed at favouring the use of French as the common language in the public sphere. Clearly, mother tongue and language spoken at home do not meet the public sphere criteria. My main argument here is that language monitoring today should be a policy-related issue and therefore aimed at measuring the evolution of the French language in the public sphere.     

Quantification of small GTPase glucosylation by clostridial glucosylating toxins using multiplexed MRM analysis

Large clostridial toxins mono‐O‐glucosylate small GTPases of the Rho and Ras subfamily. As a result of glucosylation, the GTPases are inhibited and thereby corresponding downstream signaling pathways are disturbed. Current methods for quantifying the extent of glucosylation include sequential [¹⁴C]glucosylation, sequential [³²P]ADP‐ribosylation, and Western Blot detection of nonglucosylated GTPases, with neither method allowing the quantification of the extent of glucosylation of an individual GTPase. Here, we describe a novel MS‐based multiplexed MRM assay to specifically quantify the glucosylation degree of small GTPases. This targeted proteomics approach achieves a high selectivity and reproducibility, which allows determination of the in vivo substrate pattern of glucosylating toxins. As proof of principle, GTPase glucosylation was analyzed in CaCo‐2 cells treated with TcdA, and glucosylation kinetics were determined for RhoA/B, RhoC, RhoG, Ral, Rap1, Rap2, (H/K/N)Ras, and R‐Ras2.     

Phloem translocation of Fe,Cu, Mn,and Zn in Ricinus seedlings in relation to the concentrations of nicotianamine,an endogenous chelator of divalent metal ions,in different seedling parts

During the first 8 days of germination the Ricinus seedling is supplied with all nutrients by the endosperm via phloem transport. In 4- to 8-days-old seedlings the concentrations and contents of Fe, Cu, Mn and Zn, and nicotianamine (NA) in the endosperm, cotyledons, hypocotyl and roots were estimated. From the data obtained translocation rates and flow profiles for the metals were established. The main sink for Fe, Mn and Zn were the cotyledons whereas Cu was mainly imported into the hypocotyl. Maximum flow rates occurred between days 5 and 7, for Zn between days 6 and 8.The time kinetics of NA and divalent metal ion concentrations and contents are interpreted as co-transport. The role of NA as transport vehicle of micronutrients in the sieve tubes is discussed.