Differential in vivo binding dynamics of somatic and oocyte-specific linker histones in oocytes and during ES cell nuclear transfer

The embryonic genome is formed by fusion of a maternal and a paternal genome. To accommodate the resulting diploid genome in the fertilized oocyte dramatic global genome reorganizations must occur. The higher order structure of chromatin in vivo is critically dependent on architectural chromatin proteins, with the family of linker histone proteins among the most critical structural determinants. Although somatic cells contain numerous linker histone variants, only one, H1FOO, is present in mouse oocytes. Upon fertilization H1FOO rapidly populates the introduced paternal genome and replaces sperm-specific histone-like proteins. The same dynamic replacement occurs upon introduction of a nucleus during somatic cell nuclear transfer. To understand the molecular basis of this dynamic histone replacement process, we compared the localization and binding dynamics of somatic H1 and oocyte-specific H1FOO and identified the molecular determinants of binding to either oocyte or somatic chromatin in living cells. We find that although both histones associate readily with chromatin in nuclei of somatic cells, only H1FOO is capable of correct chromatin association in the germinal vesicle stage oocyte nuclei. This specificity is generated by the N-terminal and globular domains of H1FOO. Measurement of in vivo binding properties of the H1 variants suggest that H1FOO binds chromatin more tightly than somatic linker histones. We provide evidence that both the binding properties of linker histones as well as additional, active processes contribute to the replacement of somatic histones with H1FOO during nuclear transfer. These results provide the first mechanistic insights into the crucial step of linker histone replacement as it occurs during fertilization and somatic cell nuclear transfer.     

The Clostridium botulinum GerAB germination protein is located in the inner membrane of spores

Clostridium botulinum dormant spores germinate in presence of l-alanine via a specific receptor composed of GerAA, GerAB and GerAC proteins. In Bacillus subtilis spores, GerAA and GerAC proteins were located in the inner membrane of the spore. We studied the location of the GerAB protein in C. botulinum spore fractions by Western-blot analysis, using an antipeptidic antibody. The protein GerAB was in vitro translated and used to confirm the specificity of the antibodies. GerAB was not present in a coat and spore outer membrane fraction but was present in a fraction of decoated spores containing inner membrane. These results strongly suggest that the protein GerAB is located in the inner membrane of the spore.     

Length, orientation, and plant host influence the mutation frequency in microsatellites.

Microsatellites are simple, tandem DNA repeats that represent unstable regions of the genome. They undergo frequent changes in tract length by base additions or deletions due to DNA polymerase slippage during replication. To characterize factors affecting the frequency of spontaneous mutations occurring in microsatellites in plants, a reporter system was used in Arabidopsis thaliana and tomato (Lycopersicon esculentum). The beta-glucuronidase (GUS) reporter system was used to measure the mutation frequency in various microsatellites (G(7), G(10), G(13), G(16), and C(16)) in somatic tissues. Our results indicate that this frequency increases with the number of repeats: a G(16) tract was almost 80-fold more mutable than a G(7) tract. Furthermore, the frequency of mutations depends on repeat orientation, as G(16) was 3-fold more mutable than C(16). The mutation rate was also found to differ markedly in Arabidopsis and tomato for an identical microsatellite. Indeed, Arabidopsis showed a 5-fold higher mutation frequency than tomato with the same G(7) reporter construct. Finally, mutation in a G(16) tract was frequent enough that mutations transmitted germinally to the next generation could be detected at a relatively high frequency.     

Heme induces significant neutrophil adhesion in vitro via an NFκB and reactive oxygen species-dependent pathway

Molecular and Cellular Biochemistry - Intravascular hemolysis, a major manifestation of sickle cell disease (SCD) and other diseases, incurs the release of hemoglobin and heme from red blood cells,...     

Mass Spectrometry Detection of G3m and IGHG3 Alleles and Follow-Up of Differential Mother and Neonate IgG3

Mass spectrometry (MS) analysis for detection of immunoglobulins (IG) of the human IgG3 subclass is described that relies on polymorphic amino acids of the heavy gamma3 chains. IgG3 is the most polymorphic human IgG subclass with thirteen G3m allotypes located on the constant CH2 and CH3 domains of the gamma3 chain, the combination of which leads to six major G3m alleles. Amino acid changes resulting of extensive sequencing previously led to the definition of 19 IGHG3 alleles that have been correlated to the G3m alleles. As a proof of concept, MS proteotypic peptides were defined which encompass discriminatory amino acids for the identification of the G3m and IGHG3 alleles. Plasma samples originating from ten individuals either homozygous or heterozygous for different G3m alleles, and including one mother and her baby (drawn sequentially from birth to 9 months of age), were analyzed. Total IgG3 were purified using affinity chromatography and then digested by a combination of AspN and trypsin proteases, and peptides of interest were detected by mass spectrometry. The sensitivity of the method was assessed by mixing variable amounts of two plasma samples bearing distinct G3m allotypes. A label-free approach using the high-performance liquid chromatography (HPLC) retention time of peptides and their MS mass analyzer peak intensity gave semi-quantitative information. Quantification was realized by selected reaction monitoring (SRM) using synthetic peptides as internal standards. The possibility offered by this new methodology to detect and quantify neo-synthesized IgG in newborns will improve knowledge on the first acquisition of antibodies in infants and constitutes a promising diagnostic tool for vertically-transmitted diseases.     

Layer-specific strain analysis in patients with suspected stable angina pectoris and apparently normal left ventricular wall motion

Background

Non-invasive imaging tests are widely used in the evaluation of stable angina pectoris (SAP). Despite these tests, non-significant coronary lesions are not a rare finding in patients undergoing elective coronary angiography (CAG). Two-dimensional (2D) speckle tracking global longitudinal strain (GLS) imaging is a more sensitive and accurate technique for measuring LV function than conventional 2D methods. Layer-specific strain analysis is a relatively new method that provides endocardial and epicardial myocardial layer assessment. The aim of the present study was to evaluate longitudinal layer-specific strain (LSS) imaging in patients with suspected SAP.

Methods

Patients who underwent CAG for SAP were retrospectively screened. A total of 79 patients with no history of heart disease and wall motion abnormalities were included in the study. Forty-three patients with coronary lesions >?70% constituted the coronary artery disease (CAD) group and 36 patients without significant CAD constituted the control group. Layer-specific GLS transmural, endocardium, and epicardium values (GLS-trans, GLS-endo, and GLS-epi, respectively) were compared between the groups.

Results

Patients in the CAD group had significantly lower GLS values in all layers (GLS-trans: -18.2 + 2.4% vs -22.2 + 2.2% p?<?.001; GLS-endo: -20.8 + 2.8% vs -25.3 + 2.6%, p?<?.001; GLS-epi: 15.9 + 2.4% vs -19.5 + 1.9%, p?<?.001). Multivariate adjustment demonstrated GLS-trans as the only independent predictor of CAD [OR:0.472, CI (0.326–0.684), p?<?.001]. Additionally, the GLS values were all lower in myocardial perfusion scintigraphy (MPS) true-positive patients compared with MPS false-positive patients (GLS-trans: -17.7?±?2.4 vs. -21.9?±?2.4%, p?<?.001; GLS-endo: -20.2?±?2.9% vs -24.9?±?2.9%, P?<?.001; GLS-epi: 15.4?±?2.6% vs. -19.2?±?1.8%, P?<?.001).

Conclusion

Resting layer-specific strain as assessed by 2D speckle tracking analysis demonstrated that GLS values were reduced in all layers of myocardium with SAP and with no wall motion abnormalities. LSS analysis can improve the identification of patients with significant CAD but further prospective larger scale studies are needed to put forth the incremental value of LSS analysis over transmural GLS.

     

Is Clinical Practice Concordant with the Changes in Guidelines for Antiretroviral Therapy Initiation during Primary and Chronic HIV-1 Infection? The ANRS PRIMO and COPANA Cohorts

Objective

Guidelines for initiating HIV treatment are regularly revised. We explored how physicians in France have applied these evolving guidelines for ART initiation over the last decade in two different situations: chronic (CHI) and primary HIV-1 infection (PHI), since specific recommendations for PHI are also provided in France.

Methods

Data came from the ANRS PRIMO (1267 patients enrolled during PHI in 1996–2010) and COPANA (800 subjects enrolled at HIV diagnosis in 2004–2008) cohorts. We defined as guidelines-inconsistent during PHI and CHI, patients meeting criteria for ART initiation and not treated in the following month and during the next 6 months, respectively.

Results

ART initiation during PHI dramatically decreased from 91% of patients in 1996–99 to 22% in 2007 and increased to 60% in 2010, following changes in recommendations. In 2007, however, after the CD4 count threshold was raised to 350 cells/mm³ in 2006, only 55% of the patients with CD4≤350 were treated and 66% in 2008. During CHI, ART was more frequently initiated in patients who met the criteria at entry (96%) than during follow-up: 83% when recommendation to treat was 200 and 73% when it was 350 cells/mm³. Independent risk factors for not being treated during CHI despite meeting the criteria were lower viral load, lower educational level, and poorer living conditions.

Conclusion

HIV ART initiation guidelines are largely followed by practitioners in France. What can still be improved, however, is time to treat when CD4 cell counts reach the threshold to treat. Risk factors for lack of timely treatment highlight the need to understand better how patients’ living conditions and physicians’ perceptions influence the decision to initiate treatment.