Prevention of polyglutamine oligomerization and neurodegeneration by the peptide inhibitor QBP1 in Drosophila

Polyglutamine (polyQ) diseases are a growing class of inherited neurodegenerative diseases including Huntington's disease, which are caused by abnormal expansions of the polyQ stretch in each unrelated disease protein. The expanded polyQ stretch is thought to confer toxic properties on the disease proteins through alteration of their conformation leading to pathogenic protein-protein interactions including oligomerization and/or aggregation. Hypothesizing that molecules with selective binding affinity to the expanded polyQ stretch may interfere with the pathogenic properties, we previously identified Polyglutamine Binding Peptide 1 (QBP1) from combinatorial peptide phage display libraries. We show here that a tandem repeat of the inhibitor peptide QBP1, (QBP1)(2), significantly suppresses polyQ aggregation and polyQ-induced neurodegeneration in the compound eye of Drosophila polyQ disease models, which express the expanded polyQ protein under the eye specific promoter. Most importantly, (QBP1)(2) expression dramatically rescues premature death of flies expressing the expanded polyQ protein in the nervous system, resulting in the dramatic increase of the median life span from 5.5 to 52 days. These results suggest that QBP1 can prevent polyQ-induced neurodegeneration in vivo. We propose that QBP1 prevents polyQ oligomerization and/or aggregation either by altering the toxic conformation of the expanded polyQ stretch, or by simply competing with the expanded polyQ stretches for binding to other expanded polyQ proteins. The peptide inhibitor QBP1 is a promising candidate with great potential as a therapeutic molecule against the currently untreatable polyQ diseases.     

Receptor-binding properties of modern human influenza viruses primarily isolated in Vero and MDCK cells and chicken embryonated eggs

To study the receptor specificity of modern human influenza H1N1 and H3N2 viruses, the analogs of natural receptors, namely sialyloligosaccharides conjugated with high molecular weight (about 1500 kDa) polyacrylamide as biotinylated and label-free probes, have been used. Viruses isolated from clinical specimens were grown in African green monkey kidney (Vero) or Madin-Darby canine kidney (MDCK) cells and chicken embryonated eggs. All Vero-derived viruses had hemagglutinin (HA) sequences indistinguishable from original viruses present in clinical samples, but HAs of three of seven tested MDCK-derived isolates had one or two amino acid substitutions. Despite these host-dependent mutations and differences in the structure of HA molecules of individual strains, all studied Vero- and MDCK-isolated viruses bound to Neu5Ac alpha2-6Galbeta1-4GlcNAc (6'SLN) essentially stronger than to Neu5Acalpha2-6Galbeta1-4Glc (6'SL). Such receptor-binding specificity has been typical for earlier isolated H1N1 human influenza viruses, but there is a new property of H3N2 viruses that has been circulating in the human population during recent years. Propagation of human viruses in chicken embryonated eggs resulted in a selection of variants with amino acid substitutions near the HA receptor-binding site, namely Gln226Arg or Asp225Gly for H1N1 viruses and Leu194Ile and Arg220Ser for H3N2 viruses. These HA mutations disturb the observed strict 6'SLN specificity of recent human influenza viruses.     

Induction of interleukin 2 (IL2) and interferon-γ and enhancement of IL2 receptor expression by a CD26 monoclonal antibody

The ability of the 134-2C2 monoclonal antibody (mAb; CD26) to transmit an activation signal and to affect T cell proliferation has been studied. The 134-2C2 mAb, although not being mitogenic by itself, is able to increase the proliferation of purified T cells in the presence of exogenous interleukin 2 (IL2) or phorbol 12-myristate 13-acetate (PMA). No effect of our mAb was observed on the proliferation of T cells induced by other stimuli such as Sepharose-bound CD3 mAb, phytohemagglutinin or calcium ionophore. Since the co-stimulatory effect of 134-2C2 mAb on PMA-induced T cell proliferation was strongly inhibited by an anti-Tac antibody, its involvement on the IL2/IL2 receptor pathway was investigated. An increased IL2 secretion in T cells cultured with PMA plus 134-2C2 mAb was observed and Northern blot analysis showed that the mAb 134-2C2 acts synergistically with PMA favoring the induction of both IL2 and interferon-γ mRNA synthesis, as well as the enhancement of IL2 receptor and transferrin receptor mRNA expression. Studies on mechanisms implicated in signal transduction showed that 134-2C2 mAb modifies neither intracellular calcium levels nor phosphoinositide breakdown. Additionally, no effect was exerted on protein kinase C translocation. These data suggest that the CD26 antigen is involved in T cell activation in an IL2/IL2 receptor-dependent pathway.     

Domains of macaque DC-SIGN essential for capture and transfer of simian immunodeficiency virus

The C-type lectin DC-SIGN mediates the capture and transfer of simian immunodeficiency virus (SIV) from macaque dendritic cells (DCs) to permissive T-cells. To further identify the determinants in macaque DC-SIGN required for capture and transfer of virus, we created mutants containing deletions or point mutations in the extracellular domains, and tested their ability to capture and transmit SIV. We found that SIV bound to the carbohydrate recognition domain (CRD) of macaque DC-SIGN via the envelope protein. In addition, deleting the C-terminal half of the CRD, or mutating amino acids within this region that contact Ca(2+) or mannose, disrupted virion capture activity. However, an N-terminal CRD deletion mutant was capable of binding SIV, indicating that this region was not necessary for binding. Finally, deletion of the neck domain also reduced the capacity for macaque DC-SIGN to capture SIV. Interestingly, ICAM-3, the cellular ligand for DC-SIGN, did not bind to any of the DC-SIGN mutants, including mutants with amino acid changes in the N-terminal region of the CRD. These data suggest that the binding sites for SIV and ICAM-3 may be distinct but overlapping. Together, the data demonstrate the importance of both the neck and the CRD of macaque DC-SIGN for efficient capture of SIV and binding to ICAM-3.     

A locus for posterior polymorphous corneal dystrophy (PPCD3) maps to chromosome 10

Posterior polymorphous corneal dystrophy (PPCD) is an autosomal dominant disorder characterized by corneal endothelial abnormalities, which can lead to blindness due to loss of corneal transparency and sometimes glaucoma. We mapped a new locus responsible for PPCD in a family in which we excluded the previously reported PPCD locus on 20q11, and the region containing COL8A2 on chromosome 1. Results of a 317-marker genome scan provided significant evidence of linkage of PPCD to markers on chromosome 10, with single-point LOD scores of 2.63, 1.63, and 3.19 for markers D10S208 (at (circumflex)theta = 0.03), D10S1780 (at (circumflex)theta = 0.00), and D10S578 (at (circumflex)theta = 0.06). A maximum multi-point LOD score of 4.35 was found at marker D10S1780. Affected family members shared a haplotype in an 8.55 cM critical interval that was bounded by markers D10S213 and D10S578. Our finding of another PPCD locus, PPCD3, on chromosome 10 indicates that PPCD is genetically heterogeneous. Guttae, a common corneal finding sometimes observed along with PPCD, were found among both affected and unaffected members of the proband's sib ship, but were absent in the younger generations of the family. Evaluation of phenotypic differences between family members sharing the same affected haplotype raises questions about whether differences in disease severity, including differences in response to surgical interventions, could be due to genetic background or other factors independent of the PPCD3 locus.     

Lectin histochemistry of the lymphoid organs of the chicken

Cellular interactions within the immune system are in part mediated via the carbohydrate-rich coat of the cell membrane, the glycocalyx, of which the terminal carbohydrate residues are of particular functional importance. Thus, these carbohydrate residues from thymus, bursa of Fabricius, spleen and bone marrow of 2- and 30-day-old chickens were investigated by lectin histochemistry. In the thymus, mannose as well as N-acetyl-glucosamine (glcNAc)-specific lectins labelled macrophages, epithelial reticulum cells and lymphocytes within the cortex. In the bursa of Fabricius, the brush border of the lining epithelium, the macrophages and the endothelium were labelled by mannose-specific lectins. The follicle-associated epithelium was labelled by a broad spectrum of lectins. Epithelial cells that separated the cortex from the medulla and large mononuclear cells in the cortex were only being labelled by N-acetyl-galactosamine (galNAc)-specific and glcNAc-specific lectins, respectively. In the spleen, lymphocytes of the peri-ellipsoid lymphocyte sheaths and macrophages of the red pulp were labelled by lectins of nearly all sugar specificities. In general, glycotopes of these organs were more intensively labelled in the 2-day-old chicken than in the 30-day-old chicken, indicating changes in glycotope expression during post-hatching development. Thus, cells of the avian immune system are as rich and diverse in their lectin binding sites as their mammalian counterparts, indicating that similar carbohydrate lectin interactions between cells and matrices take place in birds as well.     

Formation of 8-oxoguanine in cellular DNA of Escherichia coli strains defective in different antioxidant defences

This paper examines the relationship in Escherichia coli betweenthe in vivo content of 8-oxoguanine (8-oxoG) in chromosomalDNA and deficiencies of various key antioxidant defences. Thestructural genes for catalases (katG and katE), cytosolic superoxidedismutases (sodA and sodB) or formamidopyrimidine-DNA glycosylase(fpg) were inactivated to obtain bacterial strains lacking thescavenger enzymes for H₂O₂ or O₂·^– or the DNA repairprotein for 8-oxoG. Wild-type bacteria showed 5-fold increasedsensitivity to both lethality and mutagenesis by H₂O₂ in K medium(1 % casamino acids and 1 % glucose), as compared with nutrientbroth. This higher sensitivity was associated with increasedchromosomal oxidative damage, estimated as the 8-oxodG content,and with a marked decrease in both catalase and SOD activities.Bacteria lacking both cytosolic SODs (sodA sodB mutant) displayedincreased 8-oxodG content in chromosomal DNA (2.8-fold thatof the wild-type) when grown under standard aerated conditions.Comparatively, no significant difference in 8-oxodG contentwas observed in cells grown without aeration. Bacteria totallydevoid of catalase activity (katG katE mutant) showed wild-typecontents of 8-oxodG in chromosomal DNA when grown under aeratedconditions. Nevertheless, the protective role of catalase inpreventing formation of 8-oxodG in chromosomal DNA became evidentunder oxidative stress conditions: growth under hyperoxygenationand, particularly, following H₂O₂ exposure. Catalase deficiencyresulted in a dramatic decrease in viability after H₂O₂ exposure.A deficiency of Fpg protein also sensitized E.coli to H₂O₂ lethality,though to lesser extent than a deficiency of catalase activity.However, the scavenger enzyme and the DNA repair protein protectedequally against 8-oxoG formed in vivo upon H₂O₂ treatment. ¹To whom correspondence should be addressed. Tel: 57 218695; Fax: 57 218688; Email: bblpucuc{at}uco.es     

Concentrations of Soluble CD95 and CD8 Antigens in the Plasma and Levels of CD8+CD95+, CD8+CD38+, and CD4+CD95+ T Cells Are Markers for HIV-1 Infection and Clinical Status

Apoptosis mediated via the CD95 (FAS/APO-1) receptor is thought to play a role in the depletion of CD4+ T cells in HIV infection. In the present study expression of the CD95 antigen on lymphocyte subsets and the plasma level of soluble CD95 (sCD95) were determined in HIV-1-infected adults. The expression of CD95 was increased on CD8 cells in all groups of HIV+ individuals, while increased expression of CD95+ cells on CD4 cells was limited to individuals with CD4 counts of <200 mm³. The proportion of CD4+ that expressed CD95 was inversely correlated with the percentage of CD4+ PBL. The concentration of sCD95 was significantly higher in the plasma of HIV-infected individuals than in normal controls. The level of sCD95 in HIV-infected subjects showed no correlation with the percentage of PBL expressing CD95, indicating that the increased level of sCD95 did not reflect release from CD95+ PBL. The plasma sCD95 concentration was significantly correlated with the percentage of CD8+ cells and, particularly, with CD8+CD38– cells. A striking inverse correlation was found between the sCD95 plasma concentration and the proportion of CD4+CD95+ cells out of the total CD4+ population. There was no correlation between the serum level of sCD95 and that of soluble CD8 (sCD8), both of which were increased in the plasma of HIV+ individuals. Unlike the level of sCD95, the level of sCD8 in the plasma of HIV+ individuals. Unlike the level of sCD95, the level of sCD8 in the plasma of HIV+ individuals was correlated with the percentage of CD95+ and CD8+CD38+ cells. The present study indicates that plasma sCD95 may be one of the factors that regulate apoptotic death of lymphocytes in HIV infection.     

A meta-analytic review of HIV behavioral interventions for reducing sexual risk behavior of men who have sex with men

This meta-analysis examines the efficacy of international HIV prevention interventions designed to reduce sexual risk behavior of men who have sex with men (MSM). We performed a comprehensive search of published and unpublished English-language reports of HIV prevention interventions that focus on MSM and evaluated changes in risky sexual behavior or biologic outcomes related to sexual risk. Data from 33 studies described in 65 reports were available as of July 2003. Studies with insufficient data to calculate effect sizes were excluded from the meta-analysis. Interventions were associated with a significant decrease in unprotected anal intercourse (odds ratio [OR] = 0.77, 95% confidence interval [CI]: 0.65-0.92) and number of sexual partners (OR = 0.85, 95% CI: 0.61-0.94) and with a significant increase in condom use during anal intercourse (OR = 1.61, 95% CI: 1.16-2.22). Interventions successful in reducing risky sexual behavior were based on theoretic models, included interpersonal skills training, incorporated several delivery methods, and were delivered over multiple sessions spanning a minimum of 3 weeks. Behavioral interventions provide an efficacious means of HIV prevention for MSM. To the extent that proven HIV prevention interventions for MSM can be successfully replicated in community settings and adapted and tailored to different situations, the effectiveness of current HIV prevention efforts can be increased.