Genomic structure and homoeologous relationship of the two alpha-subunit genes of a heterotrimeric GTP-binding protein in tobacco.

A heterotrimeric GTP-binding protein (G protein) plays a number of important roles in the signal-transduction pathways of eukaryotic cells. The allotetraploid tobacco genome has two alpha-subunit genes, NtGA1 and NtGA2, of the heterotrimeric G protein. In this study, we determined the nucleotide sequences and the exon-intron structures of the NtGA loci in tobacco and its ancestral diploid species. The genomic sequences of the NtGA loci were interrupted by 13 introns. The sizes of most exons (12 of 14) were completely conserved among the NtGA genes and the Arabidopsis alpha-subunit gene (GPA1), but most introns (11 of 13) in the NtGA genes were longer than those in GPA1. In comparison with the genomic sequences of the NtGA orthologues of ancestral Nicotiana sylvestris and Nicotiana tomentosiformis, the tobacco NtGA1 and NtGA2 were concluded to be homoeologous and assigned to the S and T genomes, respectively. More than 300 mutations including insertions-deletions (indels) and nucleotide substitutions were found in the intron regions between the NtGA1 and NtGA2 loci, whereas the exon sequences were highly conserved among these and GPA1. The structural comparison revealed larger divergence at the NtGA2 locus than at NtGA1.     

Prednisolone dose-dependently influences inflammation and coagulation during human endotoxemia

The effects of steroids on the outcome of sepsis are dose dependent. Low doses appear to be beneficial, but high doses do not improve outcome for reasons that are insufficiently understood. The effects of steroids on systemic inflammation as a function of dose have not previously been studied in humans. To determine the effects of increasing doses of prednisolone on inflammation and coagulation in humans exposed to LPS, 32 healthy males received prednisolone orally at doses of 0, 3, 10, or 30 mg (n = 8 per group) at 2 h before i.v. injection of Escherichia coli LPS (4 ng/kg). Prednisolone dose-dependently inhibited the LPS-induced release of cytokines (TNF-alpha and IL-6) and chemokines (IL-8 and MCP-1), while enhancing the release of the anti-inflammatory cytokine IL-10. Prednisolone attenuated neutrophil activation (plasma elastase levels) and endothelial cell activation (von Willebrand factor). Most remarkably, prednisolone did not inhibit LPS-induced coagulation activation, measured by plasma concentrations of thrombin-antithrombin complexes, prothrombin fragment F1+2, and soluble tissue factor. In addition, activation of the fibrinolytic pathway (tissue-type plasminogen activator and plasmin-alpha(2)-antiplasmin complexes) was dose-dependently enhanced by prednisolone. These data indicate that prednisolone dose-dependently and differentially influences the systemic activation of different host response pathways during human endotoxemia.     

Peptide mimics of the group B meningococcal capsule induce bactericidal and protective antibodies after immunization

Neisseria meningitidis serogroup B (MenB) is a leading cause of sepsis and meningitis in children. No vaccine is available for the prevention of these infections because the group B capsular polysaccharide (CP) (MenB CP) is unable to stimulate an immune response, due to its similarity with human polysialic acid. Because the MenB CP bears both human cross-reactive and non-cross-reactive determinants, we developed immunogenic peptide mimics of the latter epitopes. Peptides were selected from phage display libraries for their ability to bind to a protective anti-MenB CP mAb. One of these peptides (designated 9M) induced marked elevations in serum bactericidal activity, but not polysialic acid cross-reacting Abs, after gene priming followed by carrier-conjugate boosting. Moreover, the occurrence of bacteremia was prevented in infant rats by administration of immune sera before MenB challenge. 9M is a promising lead candidate for the development of an effective and affordable anti-MenB vaccine.     

Toxic Effects of Intracerebral PrP Antibody Administration During the Course of BSE Infection in Mice

The absence of specific immune response is a hallmark of prion diseases. However, in vitro and in vivo experiments have provided evidence that an anti-PrP humoral response could have beneficial effects. Prophylactic passive immunization performed at the time of infection delayed or prevented disease. Nonetheless, the potential therapeutic effect of PrP antibodies administered shortly before the clinical signs has never been tested in vivo. Moreover, a recent study showed the potential toxicity of PrP antibodies administered intracerebrally. We aimed at evaluating the effect of a prolonged intracerebral anti-PrP antibody administration at the time of neuroinvasion in BSE infected Tg20 mice.Unexpectedly, despite a good penetration of the antibodies in the brain parenchyma, the treatment was not protective against the development of BSE. Instead, it led to an extensive neuronal loss, strong astrogliosis and microglial activation. Since this effect was observed after injection of anti-PrP antibodies as whole IgGs, F(ab′)₂ or Fab fragments, the toxicity was directly related to the ability of the antibodies to recognize native PrP and to the intracerebral concentration achieved, and not to the Fc portion or the divalence of the antibodies.This experiment shows that a prolonged treatment with anti-PrP antibodies by the intracerebral route can induce severe side-effects and calls for caution with regard to the use of similar approaches for late therapeutic interventions in humans.Key Words: prion, PrP, BSE, mice, passive immunization, intracerebral injection, anti PrP antibody, neurotoxicity     

Biophysical properties of quadruplexes containing two or three 8-bromodeoxyguanosine residues

A physico-chemical characterization, based on NMR and CD spectroscopy, of quadruplexes formed by the oligonucleotide d(TGGGT), where two or three Gs are substituted by 8-bromo-2'-deoxyguanosine residues (dGBr), is reported. The oligonucleotidic sequences d(TGBr GBr GT), d(TGBr GGBr T), d(TGGBr GBr T), and d(TGBr GBr GBr T) have been synthesized. Only sequences d(TGBr GGBr T) and d(TGBr GBr GT) were able to fold into a well defined quadruplex structure, and their CD profiles and thermal stabilities turned out to be very different from those observed for the natural counterpart, indicating that the 8-Br-dG residues dramatically affect the structure of the quadruplex.     

High sensitivity identification of membrane proteins by MALDI TOF-MASS spectrometry using polystyrene beads

Membrane proteins play a large variety of functions in life and represent 30% of all genomes sequenced. Due to their hydrophobic nature, they are tightly bound to their biological membrane, and detergents are always required to extract and isolate them before identification by mass spectrometry (MS). The latter, however remains difficult. Peptide mass fingerprinting methods using techniques such as MALDI-TOF MS, for example, have become an important analytical tool in the identification of proteins. However, PMF of membrane proteins is a real challenge for at least three reasons. First, membrane proteins are naturally present at low levels; second, most of the detergents strongly inhibit proteases and have deleterious effects on MALDI spectra; and third, despite the presence of detergent, membrane proteins are unstable and often aggregate. We took the mitochondrial uncoupling protein 1 (UCP1) as a model and showed that differential acetonitrile extraction of tryptic peptides combined with the use of polystirene Bio-Beads triggered high resolution of the MALDI-TOF identification of mitochondrial membrane proteins solubilized either with Triton-X100 or CHAPS detergents.     

Rapid and individual-specific glycoprofiling of the low abundance N-glycosylated protein tissue inhibitor of metalloproteinases-1

A gel-based method for a mass spectrometric site-specific glycoanalysis was developed using a recombinant glycoprotein expressed in two different cell lines. Hydrophilic interaction liquid chromatography at nanoscale level was used to enrich for glycopeptides prior to MS. The glycoprofiling was performed using matrix-assisted laser desorption/ionization MS and MS/MS. The method proved to be fast and sensitive and furthermore yielded a comprehensive site-specific glycan analysis, allowing a differentiation of the glycoprofiles of the two sources of recombinant protein, both comprising N-glycans of a highly heterogeneous nature. To test the potential of the method, tissue inhibitor of metalloproteinases-1 (TIMP-1), a secreted low abundance N-glycosylated protein and a cancer marker, was purified in an individual-specific manner from plasma of five healthy individuals using IgG depletion and immunoaffinity chromatography. The corresponding TIMP-1 glycoprofiles were determined to be highly similar, comprising mainly bi- and triantennary complex oligosaccharides. Additionally it was shown that platelet-derived TIMP-1 displayed a similar glycoprofile. This is the first study to investigate the glycosylation of naturally occurring human TIMP-1, and the high similarity of the glycoprofiles showed that individual-specific glycosylation variations of TIMP-1 are minimal. In addition, the results showed that TIMP-1 derived from platelets and plasma is similarly glycosylated. This comprehensive and rapid glycoprofiling of a low abundance glycoprotein performed in an individual-specific manner allows for future studies of glycosylated biomarkers for person-specific detection of altered glycosylation and may thus allow early detection and monitoring of diseases.     

Allele frequency‐free inference of close familial relationships from genotypes or low‐depth sequencing data

Knowledge of how individuals are related is important in many areas of research, and numerous methods for inferring pairwise relatedness from genetic data have been developed. However, the majority of these methods were not developed for situations where data are limited. Specifically, most methods rely on the availability of population allele frequencies, the relative genomic position of variants and accurate genotype data. But in studies of non‐model organisms or ancient samples, such data are not always available. Motivated by this, we present a new method for pairwise relatedness inference, which requires neither allele frequency information nor information on genomic position. Furthermore, it can be applied not only to accurate genotype data but also to low‐depth sequencing data from which genotypes cannot be accurately called. We evaluate it using data from a range of human populations and show that it can be used to infer close familial relationships with a similar accuracy as a widely used method that relies on population allele frequencies. Additionally, we show that our method is robust to SNP ascertainment and applicable to low‐depth sequencing data generated using different strategies, including resequencing and RADseq, which is important for application to a diverse range of populations and species.     

Short and Long-Term Effects of the Angiotensin II Receptor Blocker Irbesartan on Intradialytic Central Hemodynamics: A Randomized Double-Blind Placebo-Controlled One-Year Intervention Trial (the SAFIR Study)

Background and Aim

Little is known about the tolerability of antihypertensive drugs during hemodialysis treatment. The present study evaluated the use of the angiotensin II receptor blocker (ARB) irbesartan.

Design

Randomized, double-blind, placebo-controlled, one-year intervention trial.

Setting and Participants

Eighty-two hemodialysis patients with urine output >300 mL/day and dialysis vintage <1 year.

Intervention

Irbesartan/placebo 300 mg/day for 12 months administered as add-on to antihypertensive treatment using a predialytic systolic blood pressure target of 140 mmHg in all patients.

Outcomes and Measurements

Cardiac output, stroke volume, central blood volume, total peripheral resistance, mean arterial blood pressure, and frequency of intradialytic hypotension.

Results

At baseline, the groups were similar regarding age, comorbidity, blood pressure, antihypertensive medication, ultrafiltration volume, and dialysis parameters. Over the one-year period, predialytic systolic blood pressure decreased significantly, but similarly in both groups. Mean start and mean end cardiac output, stroke volume, total peripheral resistance, heart rate, and mean arterial pressure were stable and similar in the two groups, whereas central blood volume increased slightly but similarly over time. The mean hemodynamic response observed during a dialysis session was a drop in cardiac output, in stroke volume, in mean arterial pressure, and in central blood volume, whereas heart rate increased. Total peripheral resistance did not change significantly. Overall, this pattern remained stable over time in both groups and was uninfluenced by ARB treatment. The total number of intradialytic hypotensive episodes was (placebo/ARB) 50/63 (P = 0.4). Ultrafiltration volume, left ventricular mass index, plasma albumin, and change in intradialytic total peripheral resistance were significantly associated with intradialytic hypotension in a multivariate logistic regression analysis based on baseline parameters.

Conclusion

Use of the ARB irbesartan as an add-on to other antihypertensive therapy did not significantly affect intradialytic hemodynamics, neither in short nor long-term, and no significant increase in hypotensive episodes was seen.

Trial registration

Clinicaltrials.gov NCT00791830