Telomere dynamics in an immortal human cell line.

The integration of transfected plasmid DNA at the telomere of chromosome 13 in an immortalized simian virus 40-transformed human cell line provided the first opportunity to study polymorphism in the number of telomeric repeat sequences on the end of a single chromosome. Three subclones of this cell line were selected for analysis: one with a long telomere on chromosome 13, one with a short telomere, and one with such extreme polymorphism that no distinct band was discernible. Further subcloning demonstrated that telomere polymorphism resulted from both gradual changes and rapid changes that sometimes involved many kilobases. The gradual changes were due to the shortening of telomeres at a rate similar to that reported for telomeres of somatic cells without telomerase, eventually resulting in the loss of nearly all of the telomere. However, telomeres were not generally lost completely, as shown by the absence of polymorphism in the subtelomeric plasmid sequences. Instead, telomeres that were less than a few hundred base pairs in length showed a rapid, highly heterogeneous increase in size. Rapid changes in telomere length also occurred on longer telomeres. The frequency of this type of change in telomere length varied among the subclones and correlated with chromosome fusion. Therefore, the rapid changes in telomere length appeared occasionally to result in the complete loss of telomeric repeat sequences. Rapid changes in telomere length have been associated with telomere loss and chromosome instability in yeast and could be responsible for the high rate of chromosome fusion observed in many human tumor cell lines.     

Chromosomal anomalies in radiation-induced fibrosis in the pig.

R-banded karyotypes were established on fibroblasts from fibrotic tissues derived from experimental fibrosis induced in pigs, either surgically or by 64 Gy of gamma-rays from iridium-192. No chromosome aberrations were observed in the surgical fibrosis. In radiation-induced fibrosis, the high frequency of abnormal karyotypes and the frequent complexity of the chromosomal rearrangements suggest that the fibroblasts originated either from the 64-Gy area, or from the penumbra, but certainly not from non-irradiated areas. At early passages in vitro, almost all karyotypes were different, demonstrating a multiclonal origin of fibrotic tissue. At late passages (above 24), the situation was quite different, with the persistence of one or two clones only, demonstrating a strong selective pressure occurring in vitro.     

Functional interaction between PARP-1 and PARP-2 in chromosome stability and embryonic development in mouse

The DNA damage-dependent poly(ADP-ribose) polymerases, PARP-1 and PARP-2, homo- and heterodimerize and are both involved in the base excision repair (BER) pathway. Here, we report that mice carrying a targeted disruption of the PARP-2 gene are sensitive to ionizing radiation. Following alkylating agent treatment, parp-2(-/-)-derived mouse embryonic fibroblasts exhibit increased post-replicative genomic instability, G(2)/M accumulation and chromosome mis-segregation accompanying kinetochore defects. Moreover, parp-1(-/-)parp-2(-/-) double mutant mice are not viable and die at the onset of gastrulation, demonstrating that the expression of both PARP-1 and PARP-2 and/or DNA-dependent poly(ADP-ribosyl) ation is essential during early embryogenesis. Interestingly, specific female embryonic lethality is observed in parp-1(+/-)parp-2(-/-) mutants at E9.5. Meta phase analyses of E8.5 embryonic fibroblasts highlight a specific instability of the X chromosome in those females, but not in males. Together, these results support the notion that PARP-1 and PARP-2 possess both overlapping and non-redundant functions in the maintenance of genomic stability.     

First chemical synthesis of a scorpion alpha-toxin affecting sodium channels: the Aah I toxin of Androctonus australis hector.

Aah I is a 63-residue alpha-toxin isolated from the venom of the Buthidae scorpion Androctonus australis hector, which is considered to be the most dangerous species. We report here the first chemical synthesis of Aah I by the solid-phase method, using a Fmoc strategy. The synthetic toxin I (sAah I) was renatured in DMSO-Tris buffer, purified and subjected to thorough analysis and comparison with the natural toxin. The sAah I showed physico-chemical (CD spectrum, molecular mass, HPLC elution), biochemical (amino-acid composition, sequence), immunochemical and pharmacological properties similar to those of the natural toxin. The synthetic toxin was recognized by a conformation-dependent monoclonal anti-Aah I antibody, with an IC50 value close to that for the natural toxin. Following intracerebroventricular injection, the synthetic and the natural toxins were similarly lethal to mice. In voltage-clamp experiments, Na(v) 1.2 sodium channel inactivation was inhibited by the application of sAah I or of the natural toxin in a similar way. This work describes a simple protocol for the chemical synthesis of a scorpion alpha-toxin, making it possible to produce structural analogues in time.     

AUDIT,AUDIT-C,and AUDIT-3: Drinking Patterns and Screening for Harmful,Hazardous and Dependent Drinking in Katutura,Namibia

Objectives

To describe alcohol drinking patterns among participants in Katutura, Namibia, and to evaluate brief versions of the AUDIT against the full AUDIT to determine their effectiveness in detecting harmful drinking.

Methods

A cross-sectional survey was conducted in four constituencies and 639 participants, 18 years or older, completed a sociodemographic survey and the AUDIT. The effectiveness of the AUDIT-C (first three questions) and the AUDIT-3 (third question) was compared to the full AUDIT.

Results

Approximately 40% were identified as harmful, hazardous or likely dependent drinkers, with men having a higher likelihood than women (57.2% vs. 31.0%, p<.0001). Approximately 32% reported making and/or selling alcohol from home. The AUDIT-C performed best at a cutoff ≥ 3, better in men (sensitivity: 99.3%, specificity: 77.8%) than women (sensitivity: 91.7%, specificity: 77.4%). The AUDIT-3 performed poorly (maximum sensitivity: < 90%, maximum specificity: <51%). According to AUROC, the AUDIT-C performed better than the AUDIT-3.

Conclusions

A large proportion of participants met criteria for alcohol misuse, indicating a need for screening and referral for further evaluation and intervention. The AUDIT-C was almost as effective as the full AUDIT and may be easier to implement in clinical settings as a routine screening tool in resource-limited settings because of its brevity.     

Analysis of the interacting surface of maurotoxin with the voltage‐gated Shaker B K+ channel

Maurotoxin (MTX) is a 34‐residue toxin that was isolated initially from the venom of the scorpion Scorpio maurus palmatus. Unlike the other toxins of the α‐KTx6 family (Pi1, Pi4, Pi7, and HsTx1), MTX exhibits a unique disulfide bridge organization of the type C₁? C₅, C₂? C₆, C₃? C₄, and C₇? C₈ (instead of the conventional C₁? C₅, C₂? C₆, C₃? C₇, and C₄? C₈, herein referred to as Pi1‐like) that does not prevent its folding along the classic α/β scaffold of scorpion toxins. MTX_Pi1 is an MTX variant with a conventional pattern of disulfide bridging without any primary structure alteration of the toxin. Here, using MTX and/or MTX_Pi1 as models, we investigated how the type of folding influences toxin recognition of the Shaker B potassium channel. Amino acid residues of MTX that were studied for Shaker B recognition were selected on the basis of their homologous position in charybdotoxin, a three disulfide‐bridged scorpion toxin also active on this channel type. These residues favored either an MTX‐ or MTX_Pi1‐like folding. Our data indicate clearly that Lys²³ and Tyr³² (two out of ten amino acid residues studied) are the most important residues for Shaker B channel blockage by MTX. For activity on SKCa channels, the same amino acid residues also affect, directly or indirectly, the recognition of SK channels. The molecular modeling technique and computed docking indicate the existence of a correlation between the half cystine pairings of the mutated analogs and their activity on the Shaker B K⁺ channel. Overall, mutations in MTX could, or could not, change the reorganization of disulfide bridges of this molecule without affecting its α/β scaffold. However, changing of the peptide backbone (cross linking disulfide bridges from MTX‐like type vs MTX_Pi1‐like type) appears to have less impact on the molecule activity than mutation of certain key amino acids such as Lys²³ and Tyr³² in this toxin. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Soluble Melanoma Cell Adhesion Molecule (sMCAM/sCD146) Promotes Angiogenic Effects on Endothelial Progenitor Cells through Angiomotin

The melanoma cell adhesion molecule (CD146) contains a circulating proteolytic variant (sCD146), which is involved in inflammation and angiogenesis. Its circulating level is modulated in different pathologies, but its intracellular transduction pathways are still largely unknown. Using peptide pulldown and mass spectrometry, we identified angiomotin as a sCD146-associated protein in endothelial progenitor cells (EPC). Interaction between angiomotin and sCD146 was confirmed by enzyme-linked immunosorbent assay (ELISA), homogeneous time-resolved fluorescence, and binding of sCD146 on both immobilized recombinant angiomotin and angiomotin-transfected cells. Silencing angiomotin in EPC inhibited sCD146 angiogenic effects, i.e. EPC migration, proliferation, and capacity to form capillary-like structures in Matrigel. In addition, sCD146 effects were inhibited by the angiomotin inhibitor angiostatin and competition with recombinant angiomotin. Finally, binding of sCD146 on angiomotin triggered the activation of several transduction pathways that were identified by antibody array. These results delineate a novel signaling pathway where sCD146 binds to angiomotin to stimulate a proangiogenic response. This result is important to find novel target cells of sCD146 and for the development of therapeutic strategies based on EPC in the treatment of ischemic diseases.     

Large fragments of nef-protein and gp110 envelope glycoprotein from HIV-1. Synthesis, CD analysis and immunoreactivity

Chemical synthesis of large peptide fragments (from 18 to 66 amino acid residues long) of the gp110 envelope glycoprotein and of nef-protein from HIV-1 was achieved by the solid phase method. Stepwise assembling of the peptide chains was carried out automatically on 4-(oxymethyl)-phenylacetamidomethyl resin using the N-alpha-butyloxycarbonyl amino acids with benzyl-based side chain protecting groups. Two elongation protocols were used depending on the peptide chain length: a standard cycle, mainly characterized by a single coupling step (Boc-amino acid symmetrical anhydride in dimethylformamide), and an optimized one for large peptides, based on a double coupling strategy (Boc-amino acid symmetrical anhydride first in dimethylformamide, then in dichloromethane). Final cleavage of the peptide from the solid support was carried out by anhydrous hydrogen fluoride and crude peptides were purified by C18 reverse phase medium pressure liquid chromatography after molecular filtration. Characterization of the purified peptides was done by analytical HPLC, amino acid content determination, and circular dichroism analysis both in polar (H2O) and in non-polar (TFE) environments. Immunoreactivity of anti-nef positive sera from HIV-1 infected patients by ELISA with the synthetic peptides was investigated. The results showed four major antigenic regions of nef-protein and mainly the immunodominance of the N- and C-termini of the molecule. Several of these peptides should prove to be useful for both diagnosis and vaccination purposes.     

Temporal Trends in Treatment Outcomes for HIV-1 and HIV-2-Infected Adults Enrolled in C?te d'Ivoire's National Antiretroviral Therapy Program

Background

In Côte d''Ivoire during 2004–2007, numbers of ART enrollees increased from <5,000 to 36,943. Trends in nationally representative ART program outcomes have not yet been reported.

Methodology/Principal Findings

We conducted a retrospective chart review to assess trends in patient characteristics and attrition [death or loss to follow-up (LTFU)] over time, among a nationally representative sample of 3,682 adults (≥15 years) initiating ART during 2004–2007 at 34 health facilities. Among ART enrollees during 2004–2007, median age was 36, the proportion female was 67%, the proportion HIV-2-infected or dually HIV-1&2 reactive was 5%, and median baseline CD4⁺ T-cell (CD4) count was 135 cells/µL. Comparing cohorts initiating ART in 2004 with cohorts initiating ART in 2007, median baseline weight declined from 55 kg to 52 kg (p = 0.008) and the proportion weighing <45 kg increased from 17% to 22% (p = 0.014). During 2004–2007, pharmacy-based estimates of the percentage of new ART enrollees ≥95% adherent to ART declined from 74% to 60% (p = 0.026), and twelve-month retention declined from 86% to 69%, due to increases in 12-month mortality from 2%–4% and LTFU from 12%–28%. In univariate analysis, year of ART initiation was associated with increasing rates of both LTFU and mortality. Controlling for baseline CD4, weight, adherence, and other risk factors, year of ART initiation was still strongly associated with LTFU but not mortality. In multivariate analysis, weight <45 kg and adherence <95% remained strong predictors of LTFU and mortality.

Conclusions

During 2004–2007, increasing prevalence among ART enrollees of measured mortality risk factors, including weight <45 kg and ART adherence <95%, might explain increases in mortality over time. However, the association between later calendar year and increasing LTFU is not explained by risk factors evaluated in this analysis. Undocumented transfers, political instability, and patient dissatisfaction with crowded facilities might explain increasing LTFU.     

Small efficient cell-penetrating peptides derived from scorpion toxin maurocalcine

Maurocalcine is the first demonstrated example of an animal toxin peptide with efficient cell penetration properties. Although it is a highly competitive cell-penetrating peptide (CPP), its relatively large size of 33 amino acids and the presence of three internal disulfide bridges may hamper its development for in vitro and in vivo applications. Here, we demonstrate that several efficient CPPs can be derived from maurocalcine by replacing Cys residues by isosteric 2-aminobutyric acid residues and sequence truncation down to peptides of up to 9 residues in length. A surprising finding is that all of the truncated maurocalcine analogues possessed cell penetration properties, indicating that the maurocalcine is a highly specialized CPP. Careful examination of the cell penetration properties of the truncated analogues indicates that several maurocalcine-derived peptides should be of great interest for cell delivery applications where peptide size matters.