On the use of adenovirus dodecahedron as a carrier for glycoconjugate vaccines

Glycoconjugate Journal - Virus-Like Particles (VLPs) have been used as immunogenic molecules in numerous recombinant vaccines. VLPs can also serve as vaccine platform to exogenous antigens, usually...     

Apolar distal pocket mutants of yeast cytochrome c peroxidase: Hydrogen peroxide reactivity and cyanide binding of the TriAla,TriVal, and TriLeu variants

Three yeast cytochrome c peroxidase (CcP) variants with apolar distal heme pockets have been constructed. The CcP variants have Arg48, Trp51, and His52 mutated to either all alanines, CcP(triAla), all valines, CcP(triVal), or all leucines, CcP(triLeu). The triple mutants have detectable enzymatic activity at pH 6 but the activity is less than 0.02% that of wild-type CcP. The activity loss is primarily due to the decreased rate of reaction between the triple mutants and H₂O₂ compared to wild-type CcP. Spectroscopic properties and cyanide binding characteristics of the triple mutants have been investigated over the pH stability region of CcP, pH 4 to 8. The absorption spectra indicate that the CcP triple mutants have hemes that are predominantly five-coordinate, high-spin at pH 5 and six-coordinate, low-spin at pH 8. Cyanide binding to the triple mutants is biphasic indicating that the triple mutants have two slowly-exchanging conformational states with different cyanide affinities. The binding affinity for cyanide is reduced at least two orders of magnitude in the triple mutants compared to wild-type CcP and the rate of cyanide binding is reduced by four to five orders of magnitude. Correlation of the reaction rates of CcP and 12 distal pocket mutants with H₂O₂ and HCN suggests that both reactions require ionization of the reactants within the distal heme pocket allowing the anion to bind the heme iron. Distal pocket features that promote substrate ionization (basic residues involved in base-catalyzed substrate ionization or polar residues that can stabilize substrate anions) increase the overall rate of reaction with H₂O₂ and HCN while features that inhibit substrate ionization slow the reactions.     

Longitudinal Analysis of CCR5 and CXCR4 Usage in a Cohort of Antiretroviral Therapy-Na?ve Subjects with Progressive HIV-1 Subtype C Infection

HIV-1 subtype C (C-HIV) is responsible for most HIV-1 cases worldwide. Although the pathogenesis of C-HIV is thought to predominantly involve CCR5-restricted (R5) strains, we do not have a firm understanding of how frequently CXCR4-using (X4 and R5X4) variants emerge in subjects with progressive C-HIV infection. Nor do we completely understand the molecular determinants of coreceptor switching by C-HIV variants. Here, we characterized a panel of HIV-1 envelope glycoproteins (Envs) (n = 300) cloned sequentially from plasma of 21 antiretroviral therapy (ART)-naïve subjects who experienced progression from chronic to advanced stages of C-HIV infection, and show that CXCR4-using C-HIV variants emerged in only one individual. Mutagenesis studies and structural models suggest that the evolution of R5 to X4 variants in this subject principally involved acquisition of an “Ile-Gly” insertion in the gp120 V3 loop and replacement of the V3 “Gly-Pro-Gly” crown with a “Gly-Arg-Gly” motif, but that the accumulation of additional gp120 “scaffold” mutations was required for these V3 loop changes to confer functional effects. In this context, either of the V3 loop changes could confer possible transitional R5X4 phenotypes, but when present together they completely abolished CCR5 usage and conferred the X4 phenotype. Our results show that the emergence of CXCR4-using strains is rare in this cohort of untreated individuals with advanced C-HIV infection. In the subject where X4 variants did emerge, alterations in the gp120 V3 loop were necessary but not sufficient to confer CXCR4 usage.     

Heterogeneity of 11q13 region rearrangements in laryngeal squamous cell carcinoma analyzed by microarray platforms and fluorescence in situ hybridization

We reinvestigated rearrangements occurring in region q13 of chromosome 11 aiming to: (i) describe heterogeneity of the observed structural alterations, (ii) estimate amplicon size and (iii) identify of oncogenes involved in laryngeal cancer progression as potential targets for therapy. The study included 17 cell lines derived from laryngeal cancers and 34 specimens from primary laryngeal tumors. The region 11q13 was analyzed by fluorescence in situ hybridization (FISH), array comparative genomic hybridization (aCGH) and gene expression microarray. Next, quantitative real time PCR was used for chosen genes to confirm results from aCGH and gene expression microarray. The observed pattern of aberrations allows to distinguish three ways, in which gain and amplification involving 11q13 region may occur: formation of a homogeneously staining region; breakpoints in/near 11q13, which lead to the three to sevenfold increase of the copy number of 11q13 region; the presence of additional copies of the whole chromosome 11. The minimal altered region of gain and/or amplification was limited to ~1.8 Mb (chr.11:69,395,184–71,209,568) and comprised mostly 11q13.3 band which contain 12 genes. Five, out of these genes (CCND1, ORAOV1, FADD, PPFIA1, CTTN) had higher expression levels in comparison to healthy controls. Apart from CCND1 gene, which has an established role in pathogenesis of head and neck cancers, CTTN, ORAOV1 and FADD genes appear to be oncogene-candidates in laryngeal cancers, while a function of PPFIA1 requires further studies.     

Marine mammal culling programs: review of effects on predator and prey populations

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Co-occurring grass species differ in their associated microbial community composition in a temperate native grassland

Background and aims

Specific associations exist between plant species and the soil microbial community and these associations vary between habitat types and different plant groups. However, there is evidence that the associations are highly specific. Hence, we aimed to determine the specificity of plant-microbe relationships amongst co-occurring grass species in a temperate grassland.

Methods and results

We examined the broad microbial groups of bacteria and fungi as well as a specific fungal group, the arbuscular mycorrhizal community amongst two dominant C₃ and C₄ species and one sub-dominant C₃ species using terminal restriction fragment length polymorphism (T-RFLP) analysis. We found that the two dominant species were more similar to each other in their bacterial and arbuscular mycorrhizal community composition than either was to the sub-dominant species, but not in their fungal community composition. We also found no clear evidence that those differences were directly linked to soil chemical properties.

Conclusions

Our results demonstrate that co-occurring grass species have a distinct soil microbial community and T-RFLP analysis is able to detect plant species effect on the microbial community composition on an extremely local scale, providing an insight into the differences in the response of bacterial, fungal and arbuscular mycorrhizal communities to different, but similar and co-occurring, plant species.     

p53 and Translation Attenuation Regulate Distinct Cell Cycle Checkpoints during Endoplasmic Reticulum (ER) Stress

Cell cycle checkpoints ensure that proliferation occurs only under permissive conditions, but their role in linking nutrient availability to cell division is incompletely understood. Protein folding within the endoplasmic reticulum (ER) is exquisitely sensitive to energy supply and amino acid sources because deficiencies impair luminal protein folding and consequently trigger ER stress signaling. Following ER stress, many cell types arrest within the G₁ phase, although recent studies have identified a novel ER stress G₂ checkpoint. Here, we report that ER stress affects cell cycle progression via two classes of signal: an early inhibition of protein synthesis leading to G₂ delay involving CHK1 and a later induction of G₁ arrest associated both with the induction of p53 target genes and loss of cyclin D1. We show that substitution of p53/47 for p53 impairs the ER stress G₁ checkpoint, attenuates the recovery of protein translation, and impairs induction of NOXA, a mediator of cell death. We propose that cell cycle regulation in response to ER stress comprises redundant pathways invoked sequentially first to impair G₂ progression prior to ultimate G₁ arrest.