p53-dependent control of transactivation of the Pen2 promoter by presenilins

The senile plaques found in the brains of patients with Alzheimer's disease are mainly due to the accumulation of amyloid β-peptides (Aβ) that are liberated by γ-secretase, a high molecular weight complex including presenilins, PEN-2, APH-1 and nicastrin. The depletion of each of these proteins disrupts the complex assembly into a functional protease. Here, we describe another level of regulation of this multimeric protease. The depletion of both presenilins drastically reduces Pen2 mRNA levels and its promoter transactivation. Furthermore, overexpression of presenilin-1 lowers Pen2 promoter transactivation, a phenotype abolished by a double mutation known to prevent presenilin-dependent γ-secretase activity. PEN-2 expression is decreased by depletion of β-amyloid precursor protein (APP) and increased by the APP intracellular domain (AICD). We show that AICD and APP complement for Pen2 mRNA levels in APP/APLP1-2 knockout fibroblasts. Interestingly, overexpression of presenilin-2 greatly increases Pen2 promoter transactivation. The opposite effect triggered by both presenilins was reminiscent of our previous study, which showed that these two proteins elicit antagonistic effects on p53. Therefore, we examined the contribution of p53 on Pen2 transcription. Pen2 promoter transactivation, and Pen2 mRNA and protein levels were drastically reduced in p53–/– fibroblasts. Furthermore, PEN-2 expression could be rescued by p53 complementation in p53- and APP-deficient cells. Interestingly, PEN-2 expression was also reduced in p53-deficient mouse brain. Overall, our study describes a p53-dependent regulation of PEN-2 expression by other members of the γ-secretase complex, namely presenilins

Cytosine-to-Uracil Deamination by SssI DNA Methyltransferase

The prokaryotic DNA(cytosine-5)methyltransferase M.SssI shares the specificity of eukaryotic DNA methyltransferases (CG) and is an important model and experimental tool in the study of eukaryotic DNA methylation. Previously, M.SssI was shown to be able to catalyze deamination of the target cytosine to uracil if the methyl donor S-adenosyl-methionine (SAM) was missing from the reaction. To test whether this side-activity of the enzyme can be used to distinguish between unmethylated and C5-methylated cytosines in CG dinucleotides, we re-investigated, using a sensitive genetic reversion assay, the cytosine deaminase activity of M.SssI. Confirming previous results we showed that M.SssI can deaminate cytosine to uracil in a slow reaction in the absence of SAM and that the rate of this reaction can be increased by the SAM analogue 5’-amino-5’-deoxyadenosine. We could not detect M.SssI-catalyzed deamination of C5-methylcytosine (m5C). We found conditions where the rate of M.SssI mediated C-to-U deamination was at least 100-fold higher than the rate of m5C-to-T conversion. Although this difference in reactivities suggests that the enzyme could be used to identify C5-methylated cytosines in the epigenetically important CG dinucleotides, the rate of M.SssI mediated cytosine deamination is too low to become an enzymatic alternative to the bisulfite reaction. Amino acid replacements in the presumed SAM binding pocket of M.SssI (F17S and G19D) resulted in greatly reduced methyltransferase activity. The G19D variant showed cytosine deaminase activity in E. coli, at physiological SAM concentrations. Interestingly, the C-to-U deaminase activity was also detectable in an E. coli ung+ host proficient in uracil excision repair

MethylViewer: computational analysis and editing for bisulfite sequencing and methyltransferase accessibility protocol for individual templates (MAPit) projects

Bisulfite sequencing is a widely-used technique for examining cytosine DNA methylation at nucleotide resolution along single DNA strands. Probing with cytosine DNA methyltransferases followed by bisulfite sequencing (MAPit) is an effective technique for mapping protein–DNA interactions. Here, MAPit methylation footprinting with M.CviPI, a GC methyltransferase we previously cloned and characterized, was used to probe hMLH1 chromatin in HCT116 and RKO colorectal cancer cells. Because M.CviPI-probed samples contain both CG and GC methylation, we developed a versatile, visually-intuitive program, called MethylViewer, for evaluating the bisulfite sequencing results. Uniquely, MethylViewer can simultaneously query cytosine methylation status in bisulfite-converted sequences at as many as four different user-defined motifs, e.g. CG, GC, etc., including motifs with degenerate bases. Data can also be exported for statistical analysis and as publication-quality images. Analysis of hMLH1 MAPit data with MethylViewer showed that endogenous CG methylation and accessible GC sites were both mapped on single molecules at high resolution. Disruption of positioned nucleosomes on single molecules of the PHO5 promoter was detected in budding yeast using M.CviPII, increasing the number of enzymes available for probing protein–DNA interactions. MethylViewer provides an integrated solution for primer design and rapid, accurate and detailed analysis of bisulfite sequencing or MAPit datasets from virtually any biological or biochemical system

Clinical approach for the classification of congenital uterine malformations

A more objective, accurate and non-invasive estimation of uterine morphology is nowadays feasible based on the use of modern imaging techniques. The validity of the current classification systems in effective categorization of the female genital malformations has been already challenged. A new clinical approach for the classification of uterine anomalies is proposed. Deviation from normal uterine anatomy is the basic characteristic used in analogy to the American Fertility Society classification. The embryological origin of the anomalies is used as a secondary parameter. Uterine anomalies are classified into the following classes: 0, normal uterus; I, dysmorphic uterus; II, septate uterus (absorption defect); III, dysfused uterus (fusion defect); IV, unilateral formed uterus (formation defect); V, aplastic or dysplastic uterus (formation defect); VI, for still unclassified cases. A subdivision of these main classes to further anatomical varieties with clinical significance is also presented. The new proposal has been designed taking into account the experience gained from the use of the currently available classification systems and intending to be as simple as possible, clear enough and accurate as well as open for further development. This proposal could be used as a starting point for a working group of experts in the field

Установление границ охранной зоны линейного сооружения – магистральный газопровод "НГПЗ - Парабель"

Составлено графическое описание местоположения границ зон с особыми условиями использования территорий границ охранной зоны линейного сооружения – магистральный газопровод "НГПЗ - Парабель".A graphic description of the location of the boundaries of the zones with special conditions for the use of the territories of the boundaries of the protection zone of the linear structure – "the NGPZ-Parabel" gas pipeline has been compiled

Observations and implications of liquid–liquid phase separation at high relative humidities in secondary organic material produced by <i>α</i>-pinene ozonolysis without inorganic salts

Particles consisting of secondary organic material (SOM) are abundant in the atmosphere. To predict the role of these particles in climate, visibility and atmospheric chemistry, information on particle phase state (i.e., single liquid, two liquids and solid) is needed. This paper focuses on the phase state of SOM particles free of inorganic salts produced by the ozonolysis of <i>α</i>-pinene. Phase transitions were investigated in the laboratory using optical microscopy and theoretically using a thermodynamic model at 290 K and for relative humidities ranging from  &lt;  0.5 to 100 %. In the laboratory studies, a single phase was observed from 0 to 95 % relative humidity (RH) while two liquid phases were observed above 95 % RH. For increasing RH, the mechanism of liquid–liquid phase separation (LLPS) was spinodal decomposition. The RH range over which two liquid phases were observed did not depend on the direction of RH change. In the modeling studies, the SOM took up very little water and was a single organic-rich phase at low RH values. At high RH, the SOM underwent LLPS to form an organic-rich phase and a water-rich phase, consistent with the laboratory studies. The presence of LLPS at high RH values can have consequences for the cloud condensation nuclei (CCN) activity of SOM particles. In the simulated Köhler curves for SOM particles, two local maxima were observed. Depending on the composition of the SOM, the first or second maximum can determine the critical supersaturation for activation. Recently researchers have observed inconsistencies between measured CCN properties of SOM particles and hygroscopic growth measured below water saturation (i.e., hygroscopic parameters measured below water saturation were inconsistent with hygroscopic parameters measured above water saturation). The work presented here illustrates that such inconsistencies are expected for systems with LLPS when the water uptake at subsaturated conditions represents the hygroscopicity of an organic-rich phase while the barrier for CCN activation can be determined by the second maximum in the Köhler curve when the particles are water rich