Glucuronoxylomannan of Cryptococcus neoformans exacerbates in vitro yeast cell growth by interleukin 10-dependent inhibition of CD4+ T lymphocyte responses

Glucuronoxylomannan (GXM), the major capsular polysaccharide of Cryptococcus neoformans, is the most important virulence factor of this fungus. We analyzed the molecular events related to protective immune responses against a non-encapsulated strain of C. neoformans, mediated by murine splenic CD4(+) T lymphocytes in vitro, and the impact of GXM addition upon these events. Both the lymphoproliferation of CD4(+) T cells and the control of fungus growth were dependent on B7 co-stimulation. Addition of GXM did not modify CD4(+) T cell proliferation, but exacerbated infection in cultures obtained from normal and infected hosts. GXM enhanced the secretion of IL-10 and IL-4, while it reduced the production of pro-inflammatory cytokines TNF-alpha and IFN-gamma. The blockade of IL-10 activity with neutralizing antibodies increased TNF-alpha production and reduced yeast cell growth. The findings suggest that GXM exacerbates infection by down-regulating cell-mediated protective immune response and that IL-10 is implicated in yeast evasion.     

Surface expression of heterogeneous nuclear RNA binding protein M4 on Kupffer cell relates to its function as a carcinoembryonic antigen receptor

Elevated concentrations of carcinoembryonic antigen (CEA) in the blood are associated with the development of hepatic metastases from colorectal cancers. Clearance of circulating CEA occurs through endocytosis by liver macrophages, Kupffer cells. Previously we identified heterogeneous nuclear ribonucleoproteins M4 (hnRNP M4) as a receptor (CEAR) for CEA. HnRNP M4 has two isoform proteins (p80, p76), the full-length hnRNP M4 (CEARL) and a truncated form (CEARS) with a deletion of 39 amino acids between RNA binding domains 1 and 2, generated by alternative splicing. The present study was undertaken to clarify any isoform-specific differences in terms of their function as CEA receptor and localization. We develop anti-CEAR isoform-specific antibodies and show that both CEAR splicing isoforms are expressed on the surface of Kupffer cells and can function as CEA receptor. Alternatively, in P388D1 macrophages CEARS protein has nuclear and CEARL has cytoplasmic localization. In MIP101 colon cancer and HeLa cells the CEARS protein is localized to the nucleus and CEARL to the cytoplasm. These findings imply that different functions are assigned to CEAR isoforms depending on the cell type. The search of 39 amino acids deleted region against the Prosite data base revealed the presence of N-myristylation signal PGGPGMITIP that may be involved in protein targeting to the plasma membrane. Overall, this report demonstrates that the cellular distribution, level of expression, and relative amount of CEARL and CEARS isoforms determine specificity for CEA binding and the expression of alternative spliced forms of CEAR is regulated in a tissue-specific manner.     

An<Emphasis Type="Italic"> Alu</Emphasis>-mediated rearrangement as cause of exon skipping in Hunter disease

Hunter syndrome (Mucopolysaccharidosis type II), a rare X-linked lysosomal storage disorder, results from deleterious mutations in the iduronate-2-sulfatase ( IDS) gene located on Xq27.3-q28. Partial or complete deletions and large rearrangements have been extensively reported in the IDS gene as the basis of Hunter disease. The present report, however, is the first report on a Hunter patient in which Alu-mediated recombinations are implicated. Our patient showed the skipping of exon 8 at the cDNA level, without any splice-junction defects at the genomic level, where a new large rearrangement was identified instead. This new mutant allele consisted of an extensive deletion of IDS sequence of about 3 kb, as well as an additional inserted sequence of 157 bp. Two different computer programs were necessary to elucidate the nature of the insert. NCBI-BLAST query detected a single match for 126 bp out of 157 of the fragment that aligned exactly with a specific chromosomal region, Xq25-27.1, where an AluSg sequence is adjacent to an L1. Instead, the Repeat Masker program identified only 83 bp out of 157 of the insert, which was confirmed as an AluS. The observed homology between the AluSc sequence in the IDS intron 8 and the inserted AluS element, as well as the closeness of 26 bp Alu core sequence, considered to be a recombination hotspot, made us hypothesise upon the fact that both an Alu retrotransposition and an Alu-mediated deletion underlie the disease-producing rearrangement. We, therefore, now propose a mechanism that led to the large genomic deletion causing the production of the aberrant mRNA splicing.     

Diversity of Bacillus thuringiensis strains from Latin America with insecticidal activity against different mosquito species

The characterization of selected Bacillus thuringiensis strains isolated from different Latin America countries is presented. Characterization was based on their insecticidal activity against Aedes aegypti, Culex quinquefasciatus, and Anopheles albimanus larvae, scanning electron microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and plasmid profiles as well as PCR analysis using novel general and specific primers for cry and cyt genes encoding proteins active against mosquitoes (cyt1, cyt2, cry2, cry4A, cry4B, cry10, cry11, cry17, cry19, cry24, cry25, cry27, cry29, cry30, cry32, cry39, and cry40). Strains LBIT315, LBIT348, and IB604 showed threefold higher mosquitocidal activity against A. aegypti and C. quinquefasciatus larvae than B. thuringiensis subsp. israelensis and displayed high similarities with the B. thuringiensis subsp. israelensis used in this study with regard to protein and plasmid profiles and the presence of cry genes. Strain 147-8906 has activity against A. aegypti similar to that of B. thuringiensis subsp. israelensis but has different protein and plasmid profiles. This strain, harboring cry11, cry30, cyt1, and cyt2 genes, could be relevant for future resistance management interventions. Finally, the PCR screening strategy presented here led us to identify a putative novel cry11B gene.     

5-azacytidine alters TGF-beta and BMP signaling and induces maturation in articular chondrocytes

Maintenance of the articular surface depends on the function of articular chondrocytes (ACs) which produce matrix and are constrained from undergoing the maturation program seen in growth plate chondrocytes. Only during pathologic conditions, such as in osteoarthritis, are maturational constraints lost causing recapitulation of the process that occurs during endochondral ossification. With the aim of establishing a model to identify regulatory mechanisms that suppress AC hypertrophy, we examined the capability of 5-azacytidine (Aza) to have an impact on the maturational program of these cells. Primary ACs do not spontaneously express markers of maturation and are refractory to treatment by factors that normally regulate chondrocyte maturation. However, following exposure to Aza, ACs (i) were induced to express type X collagen (colX), Indian hedgehog, and alkaline phosphatase and (ii) showed altered colX and AP expression in response to bone morphogenetic protein-2 (BMP-2), transforming growth factor-beta (TGF-beta), and parathyroid hormone-related protein (PTHrP). Since Aza unmasked responsiveness of ACs to BMP-2 and TGF-beta, we examined the effect of Aza treatment on signaling via these pathways by assessing the expression of the TGF-beta Smads (2 and 3), the BMP-2 Smads (1 and 5), and the Smad2 and 3-degrading ubiquitin E3 ligase Smurf2. Aza-treated ACs displayed less Smad2 and 3 and increased Smad1, 5, and Smurf2 protein and showed a loss of TGF-beta signaling on the P3TP-luciferase reporter. Suggesting that Aza-induction of Smurf2 may be responsible for the loss of Smad2 and 3 protein via this pathway, immunoprecipitation and metabolic labeling experiments confirmed that Aza accelerated the ubiquitination and degradation of these targets. Overall, Aza-treated ACs represent a novel model for the study of mechanisms that regulate maturational potential of articular cartilage, with the data suggesting that maturation of these cells may be due to up-regulation of Smad1 and 5 coupled with a Smurf2-dependent degradation of Smad2 and 3 and loss of TGF-beta signaling.     

1,2,4-thiadiazole: a novel Cathepsin B inhibitor

A novel class of Cathepsin B inhibitors has been developed with a 1,2,4-thiadiazole heterocycle as the thiol trapping pharmacophore. Several compounds with different dipeptide recognition sequence (i.e., P1′–P2′=Leu-Pro-OH or P2–P1=Cbz-Phe-Ala) at the C5 position and with different substituents (i.e., OMe, Ph, or COOH) at the C3 position of the 1,2,4-thiadiazole ring have been synthesized and tested for their inhibitory activities. The substituted thiadiazoles 3a–h inhibit Cat B in a time dependent, irreversible manner. A mechanism based on active-site directed inactivation of the enzyme by disulfide bond formation between the active site cysteine thiol and the sulfur atom of the heterocycle is proposed. Compound 3a (K_i=2.6 μM, k_i/K_i=5630 M⁻¹ s⁻¹) with a C3 methoxy moiety and a Leu-Pro-OH dipeptide recognition sequence, is found to be the most potent inhibitor in this series. The enhanced inhibitory potency of 3a is a consequence of its increased enzyme binding affinity (lower K_i) rather than its increased intrinsic reactivity (higher k_i). In addition, 3a is inactive against Cathepsin S, is a poor inhibitor of Cathepsin H and is >100-fold more selective for Cat B over papain.     

Men Own the Fields, Women Own the Crops: Gender and Power in the Cameroon Grassfields

Men Own the Fields, Women Own the Crops: Gender and Power in the Cameroon Grassfields. Miriam Goheen. Madison: University of Wisconsin Press, 1996. 252 pp.     

Tamplicon-7, a Novel T-Lymphotropic Vector Derived from Human Herpesvirus 7

We describe the derivation of a novel T-cell-defective virus vector employing the human herpesvirus 7 (HHV-7). The new vector, designated Tamplicon-7, replicates in CD4(+) T cells. The system is composed of a helper virus and defective virus genomes derived by the replication of the input Tamplicon vector. There are two cis-acting functions required for the replication and packaging of the defective virus genomes in the presence of the helper virus: the viral DNA replication origin and the composite cleavage and packaging signal, which directs the cleavage and packaging of defective virus genomes. Viral DNA replication is compatible with the rolling circle mechanism, producing large head-to-tail concatemers of the Tamplicon vector. Thus, in the presence of the helper virus, the replicated vectors are packaged and secreted into the medium. Furthermore, we have shown that the vector can be employed to express a foreign gene, encoding the green fluorescent protein, in the T cells infected with the HHV-7 helper virus. We predict that the Tamplicon-7 vector might be potentially useful for gene therapy of diseases affecting the human CD4(+) T cells, including autoimmune diseases, T-cell lymphomas, and AIDS.