Identification of novel and cell type enriched cofactors of the transcription activation domain of RelA (p65 NF-kappaB).

RelA (NF-kappaB) is a transcription factor inducible by distinct stimuli in many different cell types. To find new cell type specific cofactors of NF-kappaB dependent transcription, we isolated RelA transcription activation domain binding proteins from the nuclear extracts of three different cell types. Analysis by electrophoresis and liquid chromatography tandem mass spectrometry identified several novel putative molecular partners. Some were strongly enriched in the complex formed from the nuclear extracts of specific cell types.

Expression analyses of three members of the AtPHO1 family reveal differential interactions between signaling pathways involved in phosphate deficiency and the responses to auxin, cytokinin, and abscisic acid.

The PHO1 protein is involved in loading inorganic phosphate (Pi) to the root xylem. Ten genes homologous to AtPHO1 are present in the Arabidopsis thaliana (L.) Heyn genome. From this gene family, transcript levels of only AtPHO1, AtPHO1;H1 and AtPHO1;H10 were increased by Pi-deficiency. While the up-regulation of AtPHO1;H1 and AtPHO1;H10 by Pi deficiency followed the same rapid kinetics and was dependent on the PHR1 transcription factor, phosphite only strongly suppressed the expression of AtPHO1;H1 and had a minor effect on AtPHO1;H10. Addition of sucrose was found to increase transcript levels of both AtPHO1 and AtPHO1;H1 in Pi-sufficient or Pi-deficient plants, but to suppress AtPHO1:H10 under the same conditions. Treatments of plants with auxin or cytokinin had contrasting effect depending on the gene and on the Pi status of the plants. Thus, while both hormones down-regulated expression of AtPHO1 independently of the plant Pi status, auxin and cytokinin up-regulated AtPHO1;H1 and AtPHO1;H10 expression in Pi-sufficient plants and down-regulated expression in Pi-deficient plants. Treatments with abscisic acid inhibited AtPHO1 and AtPHO1;H1 expression in both Pi-sufficient and Pi-deficient plants, but increased AtPHO1;H10 expression under the same conditions. The inhibition of expression by abscisic acid of AtPHO1 and AtPHO1;H1, and of the Pi-starvation responsive genes AtPHT1;1 and AtIPS1, was dependant on the ABI1 type 2C protein phosphatase. These results reveal that various levels of cross talk between the signal transduction pathways to Pi, sucrose and phytohormones are involved in the regulation of expression of the three AtPHO1 homologues.

Follow-up of optic pathway gliomas in children with neurofibromatosis type 1.

Optic pathway gliomas (OPG) are found in about 15% of patients with neurofibromatosis Type 1 (NF-1). The natural history of OPG is not yet well documented. Treatment in cases with growing tumors is still controversial. Twenty-one patients with NF-1 and OPG, diagnosed over a 20-year period, and followed neuroradiologically and ophthalmologically for at least two years, were reevaluated. The diagnosis of OPG was made at a mean age of 7.1 years (range 0-14.5 years); six children were asymptomatic, 15 were symptomatic. The mean follow-up was 9.0 years (2.0-18.5 (years). In eight initially operated or biopsied patients (three optic nerve and five chiasmal gliomas) tumor regrowth was found in one patient without progression on subsequent follow-up. Improvement of visual acuity occurred in one child after operation of a large suprasellar tumor and deterioration in one patient after biopsy of a chiasmal glioma. The neuroradiological follow-up of the 13 not-operated and not-radiated patients (four optic nerve and nine chiasmal gliomas) was stable in 10, progressive in three, resulting in visual loss in one patient. In 11 children (52%) a second tumor outside the optic pathway was found at a mean age of 4.0 years after the diagnosis of an OPG. Until now they are mostly asymptomatic. Second site tumors were operated in two children because of rapid tumor growth, one child died of a brainstem tumor. OPG are a frequent complication in children with NF-1, appearing within the first decade.(ABSTRACT TRUNCATED AT 250 WORDS)

Renal endothelin receptor type B upregulation in rats with low or high renin hypertension.

OBJECTIVES: To evaluate the role of endothelin-1 (ET-1) in hypertension, we investigated density and distribution of ETA and ETB receptors in hearts and kidneys of deoxycorticosterone acetate (DOCA)-salt and 1 kidney -- 1 clip (1K1C) hypertensive rats. METHODS: Five groups of uninephrectomized Wistar rats were put on a low salt diet. Three groups of rats drank tap water and two groups received saline. One group of each regimen received DOCA subcutaneously and two corresponding groups without DOCA served as controls. The fifth group of rats had the renal artery clipped to induce 1K1C hypertension. At 6 weeks, mean arterial pressure (MAP) was recorded and membrane binding assays using 125I-ET-1 were carried out. RESULTS: MAP was increased from control 122 +/- 3 to 155 +/- 6 and 218 +/- 11 mmHg in DOCA-salt and 1K1C rats, respectively, and cardiac weight index was increased. ETA receptors were predominantly expressed in the heart, whereas ETB receptors were predominant in the kidney. In the kidneys, the density of the ETB receptor subtype was upregulated in DOCA-salt and 1K1C rats from 160 +/- 8 to 217 +/- 12 and 190 +/- 2 fmol/mg (P < 0.05), respectively, and ETA tended to be downregulated (P = 0.057). Plasma renin activity was decreased in DOCA-salt rats from 17 +/- 3 to 0.17 +/- 0.01 ng/ml per h and increased in 1K1C rats on low salt diet to 30 +/- 5 ng/ml per h. CONCLUSIONS: Since ETB is the predominant endothelin receptor in the kidneys, upregulation of the ETB receptor mediating vasodilation and downregulation of the ETA receptor mediating vasoconstriction would be compatible with a mainly renal counter-regulatory effect of endothelin-1 to hypertension. Both low and high renin models of hypertension may be affected.

Transient Ischemic Dilation (TID) ratio measurement during Rb-82 cardiac PET: Comparison between three different software packages.

Background: TIDratio indirectly reflects myocardial ischemia and is correlated with cardiacprognosis. We aimed at comparing the influence of three different softwarepackages for the assessment of TID using Rb-82 cardiac PET/CT. Methods: Intotal, data of 30 patients were used based on normal myocardial perfusion(SSS<3 and SRS<3) and stress myocardial blood flow 2mL/min/g)assessed by Rb-82 cardiac PET/CT. After reconstruction using 2D OSEM (2Iterations, 28 subsets), 3-D filtering (Butterworth, order=10, ωc=0.5), data were automatically processed, and then manually processed fordefining identical basal and apical limits on both stress and rest images.TIDratio were determined with Myometrix®, ECToolbox® and QGS®software packages. Comparisons used ANOVA, Student t-tests and Lin concordancetest (ρc). Results: All of the 90 processings were successfullyperformed. TID ratio were not statistically different between software packageswhen data were processed automatically (P=0.2) or manually (P=0.17). There was a slight, butsignificant relative overestimation of TID with automatic processing incomparison to manual processing using ECToolbox® (1.07 ± 0.13 vs 1.0± 0.13, P=0.001)and Myometrix® (1.07 ± 0.15 vs 1.01 ± 0.11, P=0.003) but not using QGS®(1.02 ±0.12 vs 1.05 ± 0.11, P=0.16). The best concordance was achieved between ECToolbox®and Myometrix® manual (ρc=0.67) processing.Conclusion: Using automatic or manual mode TID estimation was not significantlyinfluenced by software type. Using Myometrix® or ECToolbox®TID was significantly different between automatic and manual processing, butnot using QGS®. Software package should be account for when definingTID normal reference limits, as well as when used in multicenter studies. QGS®software seemed to be the most operator-independent software package, whileECToolbox® and Myometrix® produced the closest results.

Influence of cassette type on the DQE of CR systems.

In our recent paper by Monnin et al. [Med. Phys. 33, 411-420 (2006)], an objective analysis of the relative performance of a computed radiography (CR) system using both standard single-side (ST-VI) and prototype dual-side read (ST-BD) plates was reported. The presampled modulation transfer function (MTF), noise power spectrum (NPS), and detective quantum efficiency (DQE) for the systems were determined at three different beam qualities representative of paediatric chest radiography, at an entrance detector air kerma of 5 microGy. Experiments demonstrated that, compared to the standard single-side read system, the MTF for the dual-side read system was slightly reduced, but a significant decrease in image noise resulted in a marked increase in DQE (+40%) in the low spatial frequency range. However, the DQE improvement for the ST-BD plate decreased with increasing spatial frequency, and, at spatial frequencies above 2.2 mm(-1), the DQE of the dual-side read system was lower than that of the single-side one.

MMP13 mutations are the cause of recessive metaphyseal dysplasia, Spahr type.

Metaphyseal dysplasia, Spahr type (MDST; OMIM 250400) was described in 1961 based on the observation of four children in one family who had rickets-like metaphyseal changes but normal blood chemistry and moderate short stature. Its molecular basis and nosologic status remained unknown. We followed up on those individuals and diagnosed the disorder in an additional member of the family. We used exome sequencing to ascertain the underlying mutation and explored its consequences on three-dimensional models of the affected protein. The MDST phenotype is associated with moderate short stature and knee pain in adults, while extra-skeletal complications are not observed. The sequencing showed that MDST segregated with a c.619T>G single nucleotide transversion in MMP13. The predicted non-conservative amino acid substitution, p.Trp207Gly, disrupts a crucial hydrogen bond in the calcium-binding region of the catalytic domain of the matrix metalloproteinase, MMP13. The MDST phenotype is associated with recessive MMP13 mutations, confirming the importance of this metalloproteinase in the metaphyseal growth plate. Dominant MMP13 mutations have been associated with metaphyseal anadysplasia (OMIM 602111), while a single child homozygous for a MMP13 mutation had been previously diagnosed as "recessive metaphyseal anadysplasia," that we conclude is the same nosologic entity as MDST. Molecular confirmation of MDST allows distinction of it from dominant conditions (e.g., metaphyseal dysplasia, Schmid type; OMIM # 156500) and from more severe multi-system conditions (such as cartilage-hair hypoplasia; OMIM # 250250) and to give precise recurrence risks and prognosis. © 2014 Wiley Periodicals, Inc.

Three-dimensional black-blood cardiac magnetic resonance coronary vessel wall imaging detects positive arterial remodeling in patients with nonsignificant coronary artery disease.

BACKGROUND: Direct noninvasive visualization of the coronary vessel wall may enhance risk stratification by quantifying subclinical coronary atherosclerotic plaque burden. We sought to evaluate high-resolution black-blood 3D cardiovascular magnetic resonance (CMR) imaging for in vivo visualization of the proximal coronary artery vessel wall. METHODS AND RESULTS: Twelve adult subjects, including 6 clinically healthy subjects and 6 patients with nonsignificant coronary artery disease (10% to 50% x-ray angiographic diameter reduction) were studied with the use of a commercial 1.5 Tesla CMR scanner. Free-breathing 3D coronary vessel wall imaging was performed along the major axis of the right coronary artery with isotropic spatial resolution (1.0x1.0x1.0 mm(3)) with the use of a black-blood spiral image acquisition. The proximal vessel wall thickness and luminal diameter were objectively determined with an automated edge detection tool. The 3D CMR vessel wall scans allowed for visualization of the contiguous proximal right coronary artery in all subjects. Both mean vessel wall thickness (1.7+/-0.3 versus 1.0+/-0.2 mm) and wall area (25.4+/-6.9 versus 11.5+/-5.2 mm(2)) were significantly increased in the patients compared with the healthy subjects (both P<0.01). The lumen diameter (3.6+/-0.7 versus 3.4+/-0.5 mm, P=0.47) and lumen area (8.9+/-3.4 versus 7.9+/-3.5 mm(2), P=0.47) were similar in both groups. CONCLUSIONS: Free-breathing 3D black-blood coronary CMR with isotropic resolution identified an increased coronary vessel wall thickness with preservation of lumen size in patients with nonsignificant coronary artery disease, consistent with a "Glagov-type" outward arterial remodeling. This novel approach has the potential to quantify subclinical disease.

Heated Optical Fiber for Distributed Soil-Moisture Measurements: A Lysimeter Experiment

An Actively Heated Fiber Optics (AHFO) method to estimate soil moisture is tested and the analysis technique improved on. The measurements were performed in a lysimeter uniformly packed with loam soil with variable water content profiles. In the first meter of the soil profi le, 30 m of fiber optic cable were installed in a 12 loops coil. The metal sheath armoring the fiber cable was used as an electrical resistance heater to generate a heat pulse, and the soil response was monitored with a Distributed Temperature Sensing (DTS) system. We study the cooling following three continuous heat pulses of 120 s at 36 W m(-1) by means of long-time approximation of radial heat conduction. The soil volumetric water contents were then inferred from the estimated thermal conductivities through a specifically calibrated model relating thermal conductivity and volumetric water content. To use the pre-asymptotic data we employed a time correction that allowed the volumetric water content to be estimated with a precision of 0.01-0.035 (m(3) m(-3)). A comparison of the AHFO measurements with soil-moisture measurements obtained with calibrated capacitance-based probes gave good agreement for wetter soils [discrepancy between the two methods was less than 0.04 (m(3) m(-3))]. In the shallow drier soils, the AHFO method underestimated the volumetric water content due to the longertime required for the temperature increment to become asymptotic in less thermally conductive media [discrepancy between the two methods was larger than 0.1 (m(3) m(-3))]. The present work suggests that future applications of the AHFO method should include longer heat pulses, that longer heating and cooling events are analyzed, and, temperature increments ideally be measured with higher frequency.

International collaborative proficiency study of Human Papillomavirus type 16 serology.

We performed an international proficiency study of Human Papillomavirus (HPV) type 16 serology. A common methodology for serology based on virus-like particle (VLP) ELISA was used by 10 laboratories in 6 continents. The laboratories used the same VLP reference reagent, which was selected as the most stable, sensitive and specific VLP preparation out of VLPs donated from 5 different sources. A blinded proficiency panel consisting of 52 serum samples from women with PCR-verified HPV 16-infection, 11 control serum samples from virginal women and the WHO HPV 16 International Standard (IS) serum were distributed. The mean plus 3 standard deviations of the negative control serum samples was the most generally useful "cut-off" criterion for distinguishing positive and negative samples. Using sensitivity of at least 50% and a specificity of 100% as proficiency criteria, 6/10 laboratories were proficient. In conclusion, an international Standard Operating Procedure for HPV serology, an international reporting system in International Units (IU) and a common "cut-off" criterion have been evaluated in an international HPV serology proficiency study.

Different types of cell death are involved in brain damage of methylmalonic aciduria and glutaric aciduria type I

We previously showed that exposure of 3D organotypic rat brain cell cultures to 1mM 2-methylcitrate (2-MCA) or 3-hydroxyglutarate (3- OHGA) every 12h over three days (DIV11-DIV14) results in ammonium accumulation and cell death. The aim of this study was to define the time course (every 24h) of the observed effects. Ammonium in culture medium already increased at DIV12 staying stable on the following days under 3-OHGA exposure, while it increased consecutively up to much higher levels under 2-MCA exposure. Lactate increase and glucose decrease were observed from DIV13 and DIV14, respectively. We conclude that ammonium accumulation precedes alterations of energy metabolism. As observed by immunohistochemistry glial cells were the predominant dying cells. Immunoblotting and immunohistochemistry with cell death specific markers (caspase-3, alpha-fodrin, LC3) showed that 2-MCA exposure significantly increased apoptosis on DIV14, but did not alter autophagy or necrosis. In contrast, 3-OHGA exposure substantially increased necrosis already from DIV13, while no change was observed for apoptosis and autophagy. In conclusion, ammonium accumulation, secondary disturbance of energy metabolism and glial cell death are involved in the neuropathogenesis ofmethylmalonic aciduria and glutaric aciduria type I. Interestingly, brain cells are dying by necrosis under 3-OHGA exposure and by apoptosis under 2-MCA exposure.

Analysis of natural variants of the human immunodeficiency virus type 1 gag-pol frameshift stem-loop structure.

Human immunodeficiency virus type 1 uses ribosomal frameshifting for translation of the Gag-Pol polyprotein. Frameshift activities are thought to be tightly regulated. Analysis of gag p1 sequences from 270 plasma virions identified in 64% of the samples the occurrence of polymorphism that could lead to changes in thermodynamic stability of the stem-loop. Expression in Saccharomyces cerevisiae of p1-beta-galactosidase fusion proteins from 10 representative natural stem-loop variants and three laboratory mutant constructs (predicted the thermodynamic stability [Delta G degrees] ranging from -23.0 to -4.3 kcal/mol) identified a reduction in frameshift activity of 13 to 67% compared with constructs with the wild-type stem-loop (Delta G degrees, -23.5 kcal/mol). Viruses carrying stem-loops associated with greater than 60% reductions in frameshift activity presented profound defects in viral replication. In contrast, viruses with stem-loop structures associated with 16 to 42% reductions in frameshift efficiency displayed no significant viral replication deficit.

Three-stranded DNA structure; is this the secret of DNA homologous recognition?

A novel type of triple-stranded DNA structure was proposed by several groups to play a crucial role in homologous recognition between single- and double-stranded DNA molecules. In this still putative structure a duplex DNA was proposed to co-ordinate a homologous single strand in its major groove side. In contrast to the well-characterized pyrimidine-purine-pyrimidine triplexes in which the two like strands are antiparallel and which are restricted to poly-pyrimidine-containing stretches, the homology-specific triplexes would have like strands in parallel orientation and would not be restricted to any particular sequence provided that there is a homology between interacting DNA molecules. For many years the stereo-chemical possibility of forming homology-dependent three- or four-stranded DNA structures during the pairing stage of recombination reactions was seriously considered in published papers. However, only recently has there been a marked increase in the number of papers that have directly tested the formation of triple-stranded DNA structures during the actual pairing stage of the recombination reaction. Unfortunately the results of these tests are not totally clear cut; while some laboratories presented experimental evidence consistent with the formation of triplexes, others studying the same or very similar systems offered alternative explanations. The aim of this review is to present the current state of the central question in the mechanism of homologous recombination, namely, what kind of DNA structure is responsible for DNA homologous recognition. Is it a novel triplex structure or just a classical duplex?

Evolution of the P-type II ATPase gene family in the fungi and presence of structural genomic changes among isolates of Glomus intraradices.

BACKGROUND: The P-type II ATPase gene family encodes proteins with an important role in adaptation of the cell to variation in external K+, Ca2+ and Na2+ concentrations. The presence of P-type II gene subfamilies that are specific for certain kingdoms has been reported but was sometimes contradicted by discovery of previously unknown homologous sequences in newly sequenced genomes. Members of this gene family have been sampled in all of the fungal phyla except the arbuscular mycorrhizal fungi (AMF; phylum Glomeromycota), which are known to play a key-role in terrestrial ecosystems and to be genetically highly variable within populations. Here we used highly degenerate primers on AMF genomic DNA to increase the sampling of fungal P-Type II ATPases and to test previous predictions about their evolution. In parallel, homologous sequences of the P-type II ATPases have been used to determine the nature and amount of polymorphism that is present at these loci among isolates of Glomus intraradices harvested from the same field. RESULTS: In this study, four P-type II ATPase sub-families have been isolated from three AMF species. We show that, contrary to previous predictions, P-type IIC ATPases are present in all basal fungal taxa. Additionally, P-Type IIE ATPases should no longer be considered as exclusive to the Ascomycota and the Basidiomycota, since we also demonstrate their presence in the Zygomycota. Finally, a comparison of homologous sequences encoding P-type IID ATPases showed unexpectedly that indel mutations among coding regions, as well as specific gene duplications occur among AMF individuals within the same field. CONCLUSION: On the basis of these results we suggest that the diversification of P-Type IIC and E ATPases followed the diversification of the extant fungal phyla with independent events of gene gains and losses. Consistent with recent findings on the human genome, but at a much smaller geographic scale, we provided evidence that structural genomic changes, such as exonic indel mutations and gene duplications are less rare than previously thought and that these also occur within fungal populations.

The unsolved puzzle of neuropathogenesis in glutaric aciduria type I.

Glutaric aciduria type I (GA-I) is a cerebral organic aciduria caused by deficiency of glutaryl-Co-A dehydrogenase (GCDH). GCDH deficiency leads to accumulation of glutaric acid (GA) and 3-hydroxyglutaric acid (3-OHGA), two metabolites that are believed to be neurotoxic, in brain and body fluids. The disorder usually becomes clinically manifest during a catabolic state (e.g. intercurrent illness) with an acute encephalopathic crisis that results in striatal necrosis and in a permanent dystonic-dyskinetic movement disorder. The results of numerous in vitro and in vivo studies have pointed to three main mechanisms involved in the metabolite-mediated neuronal damage: excitotoxicity, impairment of energy metabolism and oxidative stress. There is evidence that during a metabolic crisis GA and its metabolites are produced endogenously in the CNS and accumulate because of limiting transport mechanisms across the blood-brain barrier. Despite extensive experimental work, the relative contribution of the proposed pathogenic mechanisms remains unclear and specific therapeutic approaches have yet to be developed. Here, we review the experimental evidence and try to delineate possible pathogenetic models and approaches for future studies.