Molecular mechanisms that distinguish TFIID housekeeping from regulatable SAGA promoters

An important distinction is frequently made between constitutively expressed housekeeping genes versus regulated genes. Although generally characterized by different DNA elements, chromatin architecture and cofactors, it is not known to what degree promoter classes strictly follow regulatability rules and which molecular mechanisms dictate such differences. We show that SAGA‐dominated/TATA‐box promoters are more responsive to changes in the amount of activator, even compared to TFIID/TATA‐like promoters that depend on the same activator Hsf1. Regulatability is therefore an inherent property of promoter class. Further analyses show that SAGA/TATA‐box promoters are more dynamic because TATA‐binding protein recruitment through SAGA is susceptible to removal by Mot1. In addition, the nucleosome configuration upon activator depletion shifts on SAGA/TATA‐box promoters and seems less amenable to preinitiation complex formation. The results explain the fundamental difference between housekeeping and regulatable genes, revealing an additional facet of combinatorial control: an activator can elicit a different response dependent on core promoter class.     

Wnt-3a and Dickkopf-1 stimulate neurite outgrowth in Ewing tumor cells via a Frizzled3- and c-Jun N-terminal kinase-dependent mechanism

Recombinant Wnt-3a stimulated the rapid formation of elongated processes in Ewing sarcoma family tumor (ESFT) cells that were identified as neurites. The processes stained positively for polymerized actin and microtubules as well as synapsin I and growth-associated protein 43. Inhibition of the Wnt receptor, Frizzled3 (Fzd3), with antiserum or by short interfering RNA (siRNA) markedly reduced neurite extension. Knockdown of Dishevelled-2 (Dvl-2) and Dvl-3 also suppressed neurite outgrowth. Surprisingly, disruption of the Wnt/Fzd/lipoprotein receptor-related protein (LRP) complex and the associated beta-catenin signaling by treating cells either with the Wnt antagonist Dickkopf-1 (Dkk1) or LRP5/LRP6 siRNA enhanced neuritogenesis. Neurite outgrowth induced by Dkk1 or with LRP5/LRP6 siRNA was inhibited by secreted Fzd-related protein 1, a Wnt antagonist that binds directly to Wnt. Moreover, Dkk1 stimulation of neurite outgrowth was blocked by Fzd3 siRNA. These results suggested that Dkk1 shifted endogenous Wnt activity from the beta-catenin pathway to Fzd3-mediated, noncanonical signaling that is responsible for neurite formation. In particular, c-Jun amino-terminal kinase (JNK) was important for neurite outgrowth stimulated by both Wnt-3a and Dkk1. Our data demonstrate that Fzd3, Dvl, and JNK activity mediate Wnt-dependent neurite outgrowth and that ESFT cell lines will be useful experimental models for the study of Wnt-dependent neurite extension.     

A new method for surface staining large slices of fixed brain using a copper phthalocyanine dye

Here we describe a method for gross staining of gray matter in slices of formaldehyde-fixed human brain. After protection of white matter with 4% phenol at 60°C for 5 min followed by a cold water wash, the gray matter was stained for 10-15 min at 20-25°C with 1% aqueous copper(II) phthalocyanine tetrasulfonic acid tetrasodium salt (CPTS). The staining resisted all attempts to be washed from the gray matter. Stained slices can be stored indefinitely in slightly acidified water, or plastinated as permanent dry specimens.     

Spontaneous coronary artery dissection: long stenting in a patient with polycythemia vera

Spontaneous coronary artery dissection is a rare cause of myocardial ischemia or sudden cardiac death. We describe a patient with polycythemia vera and a chronic spontaneous coronary artery dissection who was treated with successful angioplasty and long stenting.     

Thwarting of behaviour in different contexts and the gakel-call in the laying hen

Earlier studies have shown that thwarting of feeding behaviour in the laying hen is expressed through a specific vocalisation, the gakel-call. The first aim of this study was to investigate whether the effect of deprivation per se on the occurrence of gakel-calls can be distinguished from the effect of the additional frustration. Frustration is defined as the state of an animal that results from nonreward in the expectancy of reward. The second aim was to investigate whether the occurrence of gakel-calls is restricted to a food context or whether it can be regarded as an expression of frustration in general. For this purpose, 20 hens were deprived of food, water and dustbath. After deprivation at a fixed time, a cue was given and the hens were rewarded with access to food, water or dust during a 15-min session on 4 consecutive days. On the fifth day, they were thwarted in the associated behaviours by blocking the access to these commodities, after the hens had been presented the signal that previously preceded the reward. We then recorded behaviours that might reflect the state of frustration in three 15-min periods. The period "Pre-Frustration" started 15 min before "Frustration". This, in turn, was followed by the period "Post-frustration" in which the hens were rewarded again. Nesting behaviour was thwarted by blocking the access to the nest (Frustration) after a hen had reached the last stage of its prelaying behaviour.In the food, water and dustbath context, deprivation elicited gakel-calls. The additional frustration resulted in a higher number of gakel-calls in all contexts except the food context. However, together with the findings of previous experiments, the results of this study suggest that frustration, in general, is expressed through the gakel-call. Frustration in the nest context elicited more gakel-calls than the other contexts. This latter finding is discussed in the light of the occurrence of the gakel-call under natural circumstances.     

A mRNA landscape of bovine embryos after standard and MAPK-inhibited culture conditions: a comparative analysis

Background

Genes and signalling pathways involved in pluripotency have been studied extensively in mouse and human pre-implantation embryos and embryonic stem (ES) cells. The unsuccessful attempts to generate ES cell lines from other species including cattle suggests that other genes and pathways are involved in maintaining pluripotency in these species. To investigate which genes are involved in bovine pluripotency, expression profiles were generated from morula, blastocyst, trophectoderm and inner cell mass (ICM) samples using microarray analysis. As MAPK inhibition can increase the NANOG/GATA6 ratio in the inner cell mass, additionally blastocysts were cultured in the presence of a MAPK inhibitor and changes in gene expression in the inner cell mass were analysed.

Results

Between morula and blastocyst 3,774 genes were differentially expressed and the largest differences were found in blastocyst up-regulated genes. Gene ontology (GO) analysis shows lipid metabolic process as the term most enriched with genes expressed at higher levels in blastocysts. Genes with higher expression levels in morulae were enriched in the RNA processing GO term. Of the 497 differentially expressed genes comparing ICM and TE, the expression of NANOG, SOX2 and POU5F1 was increased in the ICM confirming their evolutionary preserved role in pluripotency. Several genes implicated to be involved in differentiation or fate determination were also expressed at higher levels in the ICM. Genes expressed at higher levels in the ICM were enriched in the RNA splicing and regulation of gene expression GO term. Although NANOG expression was elevated upon MAPK inhibition, SOX2 and POU5F1 expression showed little increase. Expression of other genes in the MAPK pathway including DUSP4 and SPRY4, or influenced by MAPK inhibition such as IFNT, was down-regulated.

Conclusion

The data obtained from the microarray studies provide further insight in gene expression during bovine embryonic development. They show an expression profile in pluripotent cells that indicates a pluripotent, epiblast-like state. The inability to culture ICM cells as stem cells in the presence of an inhibitor of MAPK activity together with the reported data indicates that MAPK inhibition alone is not sufficient to maintain a pluripotent character in bovine cells.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1448-x) contains supplementary material, which is available to authorized users.     

Chemical immobilization of adult female Weddell seals with tiletamine and zolazepam: effects of age, condition and stage of lactation

Background

Postweaning diarrhoea (PWD) in pigs is usually the main infectious problem of large-scale farms and is responsible for significant losses worldwide. The disease is caused mainly by enterotoxigenic E. coli (ETEC) and Shiga-toxin producing E. coli (STEC). In this study a total of 101 E. coli isolated from pigs with PWD in Slovakia were characterized using phenotypic and genotypic methods.

Results

These 101 isolates belonged to 40 O:H serotypes. However, 57% of the isolates belonged to only six serotypes (O9:H51, O147:H-, O149:H10, O163:H-, ONT:H-, and ONT:H4), including two new serotypes (O163:H- and ONT:H4) not previously found among porcine ETEC and STEC isolated in other countries. Genes for EAST1, STb, STa, LT and Stx2e toxins were identified in 64%, 46%, 26%, 20%, and 5% of isolates, respectively. PCR showed that 35% of isolates carried genes for F18 colonization factor, and further analyzed by restriction endonuclease revealed that all of them were F18ac. Genes for F4 (K88), F6 (P987), F17, F5 (K99), F41, and intimin (eae gene) adhesins were detected in 19 %, 5%, 3%, 0.9%, 0.9%, and 0.9% of the isolates, respectively. The study of genetic diversity, carried out by PFGE of 46 representative ETEC and STEC isolates, revealed 36 distinct restriction profiles clustered in eight groups. Isolates of the same serotype were placed together in the dendrogram, but high degree of polymorphism among certain serotypes was detected.

Conclusion

Seropathotype O149:H10 LT/STb/EAST1/F4 (14 isolates) was the most commonly detected followed by O163:H- EAST1/F18 (six isolates), and ONT:H4 STa/STb/Stx2e/F18 (five isolates). Interestingly, this study shows that two new serotypes (O163:H- and ONT:H4) have emerged as pig pathogens in Slovakia. Furthermore, our results show that there is a high genetic variation mainly among ETEC of O149:H10 serotype.     

Magnesium, essential for base excision repair enzymes, inhibits substrate binding of N-methylpurine-DNA glycosylase

N-Methylpurine-DNA glycosylase (MPG) initiates base excision repair in DNA by removing a wide variety of alkylated, deaminated, and lipid peroxidation-induced purine adducts. MPG activity and other DNA glycosylases do not have an absolute requirement for a cofactor. In contrast, all downstream activities of major base excision repair proteins, such as apurinic/apyrimidinic endonuclease, DNA polymerase beta, and ligases, require Mg(2+). Here we have demonstrated that Mg(2+) can be significantly inhibitory toward MPG activity depending on its concentration but independent of substrate type. The pre-steady-state kinetics suggests that Mg(2+) at high but physiologic concentrations decreases the amount of active enzyme concentrations. Steady-state inhibition kinetics showed that Mg(2+) affected K(m), but not V(max), and the inhibition could be reversed by EDTA but not by DNA. At low concentration, Mg(2+) stimulated the enzyme activity only with hypoxanthine but not ethenoadenine. Real-time binding experiments using surface plasmon resonance spectroscopy showed that the pronounced inhibition of activity was due to inhibition in substrate binding. Nonetheless, the glycosidic bond cleavage step was not affected. These results altogether suggest that Mg(2+) inhibits MPG activity by abrogating substrate binding. Because Mg(2+) is an absolute requirement for the downstream activities of the major base excision repair enzymes, it may act as a regulator for the base excision repair pathway for efficient and balanced repair of damaged bases, which are often less toxic and/or mutagenic than their subsequent repair product intermediates.