Differentiation of Chromatin during DNA Elimination in Euplotes crassus

In Euplotes crassus, most of the micronuclear genome is eliminated during formation of a transcriptionally active macronucleus. To understand how this is mediated throughout the genome, we have examined the chromatin structure of the macronucleus-destined sequences and Tec transposons, which are dispersed in 15,000 copies in the micronuclear genome and completely eliminated during formation of the macronuclear genome. Whereas the macronucleus-destined sequences show a typical pattern of nucleosomal repeats in micrococcal nuclease digests, the Tec element chromatin structure digests to a nucleosome-like repeat pattern that is not typical: the minimum digestion products are ∼300–600 base pairs, or “subnucleosomal,” in size. In addition, the excised, circular forms of the Tec elements are exceedingly resistant to nucleases. Nevertheless, an underlying nucleosomal structure of the Tec elements can be demonstrated from the size differences between repeats in partial micrococcal nuclease digests and by trypsin treatment of nuclei, which results in mononucleosome-sized products. Characterization of the most micrococcal nuclease–resistant DNA indicates that micronuclear telomeres are organized into a chromatin structure with digestion properties identical to those of the Tec elements in the developing macronucleus. Thus, these major repetitive sequence components of the micronuclear genome differ in their chromatin structure from the macronuclear-destined sequences during DNA elimination. The potential role of developmental stage–specific histone variants in this chromatin differentiation is discussed.

Cloning and Characterization of a Gibberellin-Induced RNase Expressed in Barley Aleurone Cells1

We cloned a cDNA for a gibberellin-induced ribonuclease (RNase) expressed in barley (Hordeum vulgare) aleurone and the gene for a second barley RNase expressed in leaf tissue. The protein encoded by the cDNA is unique among RNases described to date in that it contains a novel 23-amino acid insert between the C2 and C3 conserved sequences. Expression of the recombinant protein in tobacco (Nicotiana tabacum) suspension-cultured protoplasts gave an active RNase of the expected size, confirming the enzymatic activity of the protein. Analyses of hormone regulation of expression of mRNA for the aleurone RNase revealed that, like the pattern for α-amylase, mRNA levels increased in the presence of gibberellic acid, and its antagonist abscisic acid prevented this effect. Quantitative studies at early times demonstrated that cycloheximide treatment of aleurone layers increased mRNA levels 4-fold, whereas a combination of gibberellin plus cycloheximide treatment was required to increase α-amylase mRNA levels to the same extent. These results are consistent with loss of repression as an initial effect of gibberellic acid on transcription of those genes, although the regulatory pathways for the two genes may differ.

A structural model for a homeotic protein-extradenticle-DNA complex accounts for the choice of HOX protein in the heterodimer.

The genes of the homeotic complex (HOX) encode DNA binding homeodomain proteins that control developmental fates by differentially regulating the transcription of downstream target genes. Despite their unique in vivo functions, disparate HOX proteins often bind to very similar DNA sequences in vitro. Thus, a critical question is how HOX proteins select the correct sets of target genes in vivo. The homeodomain proteins encoded by the Drosophila extradenticle gene and its mammalian homologues, the pbx genes, contribute to HOX specificity by cooperatively binding to DNA with HOX proteins. For example, the HOX protein labial cooperatively binds with extradenticle protein to a 20-bp oligonucleotide that is sufficient to direct a labial-like expression pattern in Drosophila embryos. Here we have analyzed the protein-DNA interactions that are important for forming the labial-extradenticle-DNA complex. The data suggest a model in which labial and extradenticle, separated by only 4 bp, bind this DNA as a heterodimer in a head-to-tail orientation. We have confirmed several aspects of this model by characterizing extradenticle-HOX binding to mutant oligonucleotides. Most importantly, mutations in base pairs predicted to contact the HOX N-terminal arm resulted in a change in HOX preference in the heterodimer, from labial to Ultrabithorax. These results demonstrate that extradenticle prefers to bind cooperatively with different HOX proteins depending on subtle differences in the heterodimer binding site.

Age model, radiocarbon dates and dinoflagellate cyst measurements from sediment core DA06-139G

A marine sediment core from Vaigat in Disko Bugt, West Greenland, has been analysed in terms of lithology, dinoflagellate cysts and foraminifera in order to evaluate the influence of oceanographic variability on West Greenland glacier stability. The data show that during the past 5200 years the Atlantic foraminiferal abundance in the subsurface waters of the West Greenland Current (WGC) episodically increased, indicating periods of increases in the inflow of subsurface warm Atlantic water at 2000 - 1500 cal. yr BP and 1300 cal. yr BP as well as periods of less pronounced increased bottom-water temperatures around 4700 - 4000 cal. yr BP, 3100 - 2800, 2600, 1000 - 800, 500 - 400, and at 200 cal. yr. The sedimentological and dinoflagellate cyst data indicate that these episodes with enhanced advection of Irminger Sea-derived waters are accompanied by increased iceberg rafting, which we link to increased iceberg calving in relation to destabilization of the Jakobshavn Isbrae. The long-term trend in the data documents the end of a late-Holocene Thermal Maximum between 5200 and 4300 cal. yr BP and a final onset of the Neoglaciation at 3500 cal. yr BP. Increased responses of the iceberg rafting after 3500 cal. yr BP, reflects a westward/seaward advance of the glacier margin in relation to onset of Neoglaciation and a development of the glacier into a floating tongue after 2000 cal. yr BP. A comparison of our record with a record from the eastern North Atlantic indicates that a NAO-like anomaly pattern between subsurface waters in West Greenland and atmospheric temperature in the Eastern North Atlantic may have been operating during most of the late Holocene. However, during the past 1000 years the NAO signal may have weakened as some other mode of climate variability overprints the anti-phase climate signal in this region.

Putting teeth into the developmental origins hypothesis: A longitudinal study of early childhood malnutrition, enamel hypoplasia and adolescent health in Amazonian Bolivia

Thesis (Ph.D.)--University of Washington, 2016-06

A new morphological variant of uterine PEComas with sex-cord-like pattern and WT1 expression: more doubts about the existence of uterine PEComas

PEComas are rare neoplasms that are sometimes associated with the tuberous sclerosis complex. They typically contain perivascular epithelioid cells that coexpress muscle and melanocytic markers. However, apart from these classical features, considerable clinical, pathologic, and immunohistochemical variation has been reported. WT1, the Wilms tumor gene product, can be expressed in various tumors from different anatomical sites, including sex-cord and other ovarian tumors with a sertoliform pattern. Neither a sex-cord like pattern nor WT1 expression has been described in PEComas. Here, we describe a case of uterine PEComa with a pattern of infiltration into the myometrium that is similar to stromal sarcomas, characterized by tongues and endovascular growing. The architecture and cellular morphology were similar to sex-cord tumors, and the PEComa was diffusely and strongly positive for WT1. We reviewed, from our files, an additional 9 cases of PEComa from different sites, and found WT1 expression in one more soft tissue tumor. We discuss the relationship between PEComas and other uterine sarcomas. (C) 2010 Elsevier Inc. All rights reserved.

Cardiorespiratory fitness is negatively associated with metabolic risk factors independently of the adherence to a healthy dietary pattern

Background and aim: Cardiorespiratory fitness (CRF) and diet have been involved as significant factors towards the prevention of cardio-metabolic diseases. This study aimed to assess the impact of the combined associations of CRF and adherence to the Southern European Atlantic Diet (SEADiet) on the clustering of metabolic risk factors in adolescents. Methods and Results: A cross-sectional school-based study was conducted on 468 adolescents aged 15-18, from the Azorean Islands, Portugal. We measured fasting glucose, insulin, total cholesterol (TC), HDL-cholesterol, triglycerides, systolic blood pressure, waits circumference and height. HOMA, TC/HDL-C ratio and waist-to-height ratio were calculated. For each of these variables, a Z-score was computed by age and sex. A metabolic risk score (MRS) was constructed by summing the Z scores of all individual risk factors. High risk was considered when the individual had 1SD of this score. CRF was measured with the 20 m-Shuttle-Run- Test. Adherence to SEADiet was assessed with a semi-quantitative food frequency questionnaire. Logistic regression showed that, after adjusting for potential confounders, unfit adolescents with low adherence to SEADiet had the highest odds of having MRS (OR Z 9.4; 95%CI:2.6e33.3) followed by the unfit ones with high adherence to the SEADiet (OR Z 6.6; 95% CI: 1.9e22.5) when compared to those who were fit and had higher adherence to SEADiet.

Expression pattern of sirtuins in innate immune cells and influence of Sirtuin 1/2 inhibition on innate immune responses to Toll-like receptor ligands and to infection

Purpose/Objective: The family of histone deacetylases comprises 18 members in mammals, among which seven sirtuins (SIRT1-7). Sirtuins are NADP-dependent enzymes that have been involved in the control of cell metabolism, proliferation and survival. The expression pattern of sirtuins and their influence on host response to microbial infection remain largely unknown. The aim of the study was to analyze the expression of SIRT1-7 and to address the effects of SIRT1/2 inhibition on innate immune responses in vitro and in vivo.. Materials and methods: in vitro: Bone marrow (BM), BM-derived macrophages (BMDMs) and dendritic cells (BMDCs) and RAW 264.7 and J774.1 macrophage cell lines were stimulated for 0, 2, 6 and 18 h with LPS, Pam3CSK4 and CpG ODN. SIRT1-7 mRNA was quantified by real time-PCR. TNF was measured by ELISA. In vivo: BALB/c mice were challenged with LPS (350 lg i.p.) with or without a SIRT1/2 inhibitor. Blood and organs were collected after 0, 1, 4, 8 and 24 h to quantify SIRT1-7 and TNF. Mortality was assessed daily. Results: Bone marrow, macrophages and DCs express, in order of abundance, SIRT2 > > SIRT1, SIRT3 and SIRT6 > SIRT4, SIRT5 and SIRT7. Microbial products decrease the expression of all sirtuins except SIRT6 in a time dependent manner in BMDMs (0_24 h). SIRT2 is the most expressed sirtuin also in the liver, kidney (together with SIRT3) and spleen. Upon LPS challenge, SIRT1, SIRT3, SIRT4 and SIRT7 mRNA levels decrease in the liver (from 4 h to 24 h), whereas SIRT1-7 mRNA levels decrease within 1 h in both kidney and spleen. Pharmacological inhibition of SIRT1/2 decreases TNF production by macrophages stimulated with LPS, Pam3CSK4 and CpG ODN (n = 6; P < 0.001). In agreement, prophylactic treatment with a SIRT1/2 inhibitor decreases TNF production (n = 8; P = 0.04) and increases survival (n = 13, P = 0.03) of mice challenged with LPS. Conclusions: Sirtuins are expressed in innate immune cells. Inhibition of SIRT1/2 activity decreases cytokine production by macrophages and protects from endotoxemia, suggesting that sirtuin inhibitors may represent novel adjunctive therapy for treating inflammatory disorders such as sepsis.

Food consumption impacts of adherence to dietary norms in the United States: a quantitative assessment

Promotion of adherence to healthy-eating norms has become an important element of nutrition policy in the United States and other developed countries. We assess the potential consumption impacts of adherence to a set of recommended dietary norms in the United States using a mathematical programming approach. We find that adherence to recommended dietary norms would involve significant changes in diets, with large reductions in the consumption of fats and oils along with large increases in the consumption of fruits, vegetables, and cereals. Compliance with norms recommended by the World Health Organization for energy derived from sugar would involve sharp reductions in sugar intakes. We also analyze how dietary adjustments required vary across demographic groups. Most socio-demographic characteristics appear to have relatively little influence on the pattern of adjustment required to comply with norms, Income levels have little effect on required dietary adjustments. Education is the only characteristic to have a significant influence on the magnitude of adjustments required. The least educated rather than the poorest have to bear the highest burden of adjustment. Out- analysis suggests that fiscal measures like nutrient-based taxes may not be as regressive as commonly believed. Dissemination of healthy-eating norms to the less educated will be a key challenge for nutrition policy.

Food consumption impacts of adherence to dietary norms in the United States: a quantitative assessment

Promotion of adherence to healthy-eating norms has become an important element of nutrition policy in the United States and other developed countries. We assess the potential consumption impacts of adherence to a set of recommended dietary norms in the United States using a mathematical programming approach. We find that adherence to recommended dietary norms would involve significant changes in diets, with large reductions in the consumption of fats and oils along with large increases in the consumption of fruits, vegetables, and cereals. Compliance with norms recommended by the World Health Organization for energy derived from sugar would involve sharp reductions in sugar intakes. We also analyze how dietary adjustments required vary across demographic groups. Most socio-demographic characteristics appear to have relatively little influence on the pattern of adjustment required to comply with norms, Income levels have little effect on required dietary adjustments. Education is the only characteristic to have a significant influence on the magnitude of adjustments required. The least educated rather than the poorest have to bear the highest burden of adjustment. Out- analysis suggests that fiscal measures like nutrient-based taxes may not be as regressive as commonly believed. Dissemination of healthy-eating norms to the less educated will be a key challenge for nutrition policy.

A two-armed pattern in flickering maps of the nova-like variable UU aquarii

We report the analysis of a uniform sample of 31 light curves of the nova-like variable UU Aqr with eclipse-mapping techniques. The data were combined to derive eclipse maps of the average steady-light component, the long-term brightness changes, and the low- and high-frequency flickering components. The long-term variability responsible for the ""low-brightness`` and ""high-brightness`` states is explained in terms of the response of a viscous disk to changes of 20%-50% in the mass transfer rate from the donor star. Low- and high-frequency flickering maps are dominated by emission from two asymmetric arcs reminiscent of those seen in the outbursting dwarf nova IP Peg, and they are similarly interpreted as manifestations of a tidally induced spiral shock wave in the outer regions of a large accretion disk. The asymmetric arcs are also seen in the map of the steady light aside from the broad brightness distribution of a roughly steady-state disk. The arcs account for 25% of the steady-light flux and are a long-lasting feature in the accretion disk of UU Aqr. We infer an opening angle of 10 degrees +/- 3 degrees for the spiral arcs. The results suggest that the flickering in UU Aqr is caused by turbulence generated after the collision of disk gas with the density-enhanced spiral wave in the accretion disk.

Metacarpophalangeal pattern profile in Marfan syndrome and Marfan-like patients

Marfan syndrome (MFS) is an autosomal dominant trait due to mutations in the fibrillin gene (FBN1). The MFS expressivity is variable, and its diagnosis relies completely on clinical criteria. Atypical cases and Marfan- like (marfanoid) clinical presentations are commonly found. The metacarpophalangeal pattern profile (MCPP), a radiological method in which the 19 tubular hand bones are assessed, has been used in the diagnosis of various syndromes. To investigate whether the MCPP was adequate to discriminate between MFS and Marfan-like subjects, we studied 38 patients who were referred to our service because they had an MFS diagnosis, diagnostic hypothesis, or differential diagnosis or had arachnodactyly with dolichostenomelia. Two groups were formed: 1) MFS: 21 patients with a mean age of 18.3 (10.8 S.D.) years and 2) Marfan-like syndromes: 16 patients who did not meet the current criteria, with a mean age of 14.6 (4.6 S.D.) years. The MCPP was performed in each case following the classical technique, and a characteristic mean profile was obtained for group I (MFS), with Z scores ranging from 0.69 to 2.73 (1.80 ± 0.50; mean ± S.D.). In group I, three cases had no correlation with the typical MFS pattern. In group II, three cases had an MFS pattern. The correlation with the mean MCPP of MFS permitted the differential diagnosis of MFS and marfanoid syndromes with 86% sensitivity, 81% specificity, and 86% positive and 81% negative predictive values. The results suggest that MCPP can be used effectively as an auxiliary tool in the nosology of these conditions and, because there is no change in MCPP with age, can be helpful in early diagnosis.

Dissection of the role of pili and type 2 and 3 secretion systems in adherence and biofilm formation of an atypical enteropathogenic escherichia coli strain

Atypical enteropathogenic Escherichia coli (aEPEC) strains are diarrheal pathogens that lack bundle-forming pilus production but possess the virulence-associated locus of enterocyte effacement. aEPEC strain 1551-2 produces localized adherence (LA) on HeLa cells; however, its isogenic intimin (eae) mutant produces a diffuse-adherence (DA) pattern. In this study, we aimed to identify the DA-associated adhesin of the 1551-2 eae mutant. Electron microscopy of 1551-2 identified rigid rod-like pili composed of an 18-kDa protein, which was identified as the major pilin subunit of type 1 pilus (T1P) by mass spectrometry analysis. Deletion of fimA in 1551-2 affected biofilm formation but had no effect on adherence properties. Analysis of secreted proteins in supernatants of this strain identified a 150-kDa protein corresponding to SslE, a type 2 secreted protein that was recently reported to be involved in biofilm formation of rabbit and human EPEC strains. However, neither adherence nor biofilm formation was affected in a 1551-2 sslE mutant. We then investigated the role of the EspA filament associated with the type 3 secretion system (T3SS) in DA by generating a double eae espA mutant. This strain was no longer adherent, strongly suggesting that the T3SS translocon is the DA adhesin. In agreement with these results, specific anti-EspA antibodies blocked adherence of the 1551-2 eae mutant. Our data support a role for intimin in LA, for the T3SS translocon in DA, and for T1P in biofilm formation, all of which may act in concert to facilitate host intestinal colonization by aEPEC strains. ©2013, American Society for Microbiology.

Expression Pattern and Promoter Analysis of a Eucalyptus grandis Germin-like Gene

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)