22,548 research outputs found
A Conserved GA Element in TATA-Less RNA Polymerase II Promoters
No full textInitiation of RNA polymerase (Pol) II transcription requires assembly of the pre-initiation complex (PIC) at the promoter. In the classical view, PIC assembly starts with binding of the TATA box-binding protein (TBP) to the TATA box. However, a TATA box occurs in only 15% of promoters in the yeast Saccharomyces cerevisiae, posing the question how most yeast promoters nucleate PIC assembly. Here we show that one third of all yeast promoters contain a novel conserved DNA element, the GA element (GAE), that generally does not co-occur with the TATA box. The distance of the GAE to the transcription start site (TSS) resembles the distance of the TATA box to the TSS. The TATA-less TMT1 core promoter contains a GAE, recruits TBP, and supports formation of a TBP-TFIIB-DNA-complex. Mutation of the promoter region surrounding the GAE abolishes transcription in vivo and in vitro. A 32-nucleotide promoter region containing the GAE can functionally substitute for the TATA box in a TATA-containing promoter. This identifies the GAE as a conserved promoter element in TATA-less promoters
Muscarinic receptor activation modulates neurotrophic factors production in rat Schwann-like cells derived from adipose mesenchymal stem cells.
No full textMuscarinic receptor activation modulates neurotrophic factors production in rat Schwann-like cells derived from adipose mesenchymal stem cells
Piovesana R1, Faroni A2, Soligo M3, Manni L3, Reid AJ2 & Tata AM1
1Dept. Biol and Biotech. C. Darwin, University of Rome “Sapienza”, Rome, Italy; 2Blond McIndoe Lab, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK; 3Institute of Translational Pharmacology-CNR, Rome, Italy
Peripheral nerve injury is commonly caused by direct mechanical trauma. Regeneration depends on the ability of Schwann cells (SCs) to create a favourable environment, by producing neurotrophic factors. Although SCs are effective in promoting nerve regeneration, they are not a convenient source of transplantable cells to improve outcomes after injury. Mesenchymal Stem Cells derived from adipose tissue (ASCs) seem to be a promising alternative source considering their ability to differentiate towards SC phenotype (Schwann-like).
SCs express different receptors for neurotransmitters. In particular cholinergic stimulation of M2 muscarinic receptor decreases SCs proliferation whilst upregulating myelination. Previously, we demonstrated that Schwann-like cells express muscarinic receptors; in particular the M2 receptor activation resulted in decreased proliferation and reduced migration.
In present work, we have characterised the effects mediated by muscarinic receptors on neurotrophic factors (NFs) expression and production. The selective activation of M2 receptors by arecaidine propargyl ester (APE) caused a significant decrease of the transcript levels for NFs (NGF, BDNF and GDNF), while the non-selective agonist muscarine did not influence NFs mRNA expression. By custom made Elisa Assay, we analysed the production of two different NGF forms, precursor (proNGF) and mature NGF (mNGF). APE treatment induced a decreased release of both NGF forms, whereas muscarine treatment stimulated an increased release of mNGF. Western blot analysis indicated that both agonists caused a significant decrease in the expression of the proNGF isoform at 25 kDa, which is likely involved in the modulation of apoptotic processes.
The data obtained suggest a relevant role of muscarinic receptors in the modulation of NFs production in Schwann-like cells. In particular the ability of both muscarinic agonists to negatively modulate the proNGF isoform, thereby suggesting a neuroprotective role of muscarinic receptors towards regenerating axons
Analysis of a hybrid TATA box binding protein originating from mesophilic and thermophilic donor organisms
No full textThe TATA Box Binding Protein (TBP) is a 20 kD protein that is essential and universally conserved in eucarya and archaea. Especially among archaea, organisms can be found that live below 0°C as well as organisms that grow above 100°C. The archaeal TBPs show a high sequence identity and a similar structure consisting of α-helices and β-sheets that are arranged in a saddle-shape 2-symmetric fold. In previous studies, we have characterized the thermal stability of thermophilic and mesophilic archaeal TBPs by infrared spectroscopy and showed the correlation between the transition temperature (Tm) and the optimal growth temperature (OGT) of the respective donor organism. In this study, a “new” mutant TBP has been constructed, produced, purified and analyzed for a deeper understanding of the molecular mechanisms of thermoadaptation. The β-sheet part of the mutant consists of the TBP from Methanothermobacter thermoautotrophicus (OGT 65°C, MtTBP65) whose α-helices have been exchanged by those of Methanosarcina mazei (OGT 37°C, MmTBP37). The Hybrid-TBP irreversibly aggregates after thermal unfolding just like MmTBP37 and MtTBP65, but the Tm lies between that of MmTBP37 and MtTBP65 indicating that the interaction between the α-helical and β-sheet part of the TBP is crucial for the thermal stability. The temperature stability is probably encoded in the variable α-helices that interact with the highly conserved and DNA binding β-sheets
Cholinergic effects mediated by M2 muscarinic receptor in human Schwann-like cells induced from adipose mesenchymal stem cells
No full textSchwann cells (SCs) have an important role in peripheral nerve regeneration but there are several restrictions hindering their clinical application. Adipose derived stem cells (ASCs) feature good properties for cell therapies. When exposed to specific growth factors in vitro, they can acquire a SC-like phenotype (dASCs), expressing key SCs markers. Our group has demonstrated that M2 muscarinic receptor in rat dASCs causes a reversible arrest of cell proliferation, increasing SCs myelinating phenotype. Human dASCs, as rat dASCs, express muscarinic receptors. In the present work we evaluate if M2 muscarinic receptor activation may contribute to human dASCs proliferation, migration and phenotype. M2 selective activation by selective agonist Arecaidine Propargyl Ester (APE) causes a decreased cell proliferation, modulating the expression of genes involved in the proliferation/differentiation (i.e. c-jun and egr2) and several neurotrophic factors. Moreover, M2 selective activation is able to decrease cell migration. Although further analyses are needed to best characterise the role of M2 receptor, these results are the first evidence that its selective activation may have effects also on human dASCs proliferation and migration. This may improve our knowledge of these promising therapeutic cells for potential use in nerve regeneration
Muscarinic receptor in Schwann-like adipose derived stem cells: implications in nerve regeneration.
No full textSchwann Cells (SCs) play a central role in the physiology and in the response of the axon injury. The capacity of SCs to proliferate, to secrete growth factors, to modulate immune response, to migrate and to re-myelinate regenerating nerves have been reported (Jessen et al, 2016). However, SCs present limited clinical application, such as the difficulty in collection and culture and the slow rate of in vitro expansion.
Some papers describe that Adipose-derived stem cells (ASCs) have the ability to differentiate towards SCs phenotype (Schwann-like, dASC) following exposure to suitable culture media (Kingham et al, 2007). dASC, like SCs, express functional receptors for different neurotrasmitters, including muscarinic receptor subtypes (M1-M4) that regulate some physiological events.
In the present work, we have characterised the effects mediated by muscarinic receptors on proliferation and neurotrophic factors (NFs) expression and production
Mechanical characterization of AISI 316 tubes filled with Al alloy foams
No full textIn tubular elements filled with metal foams the structural collapse is delayed in comparison with the empty tubes, consequently compression strength and absorbed energy increase. Production methods of foams are crucial, as they determine cellular morphology and bonds formation with tube wall. In this work AISI 316 steel tubes filled with foam of commercially pure Al and Al-Si alloys with hypoeutectic compositions were produced. The process parameters were optimized to obtain closed cells foams with an acceptable morphology of porosity and good mechanical properties. Foams were characterized by optical and scanning electron microscopy and by computer tomography; mechanical properties were investigated by axial compression tests (performed on foam samples and on Cu tubes, empty or filled with foams) and radial compression "Brazilian test" (carried out on AISI 316 tubes, empty or filled with foams)
A Small Trinucleotide Expansion in the TBP Gene Gives Rise to a Sporadic Case of SCA17 with Abnormal Putaminal Findings on MRI
No full textA Japanese woman developed gait disturbances at 25 years of age, and subsequently underwent gradual changes in her personality. By the age of 42, she showed clear signs of dementia and cerebellar ataxia, and displayed behavioral abnormalities, choreic movements and hyperreflexia. The findings of MRI not only showed cerebellar and cerebral atrophy, but also revealed putaminal rim hyperintensity on T2-weighted images. We identified a heterozygously expanded CAG/CAA repeat (45/36) within the TATA-binding protein gene, leading to a diagnosis of SCA17. These results show that a 45 CAG/CAA repeat is pathological, giving rise to early-onset SCA17.</p
A structural investigation of bacterial twin-arginine translocation (tat) complexes by single-particle electron microscopy
No full textThe Twin arginine translocase (Tat) pathway was first characterised in chloroplast
thylakoid membranes in the late 1990s. It has since been identified in the plasma
membranes of both Gram-positive and Gram-negative bacteria. Substrates of this
transport system contain a critical twin-arginine motif within their cleavable Nterminal
signal sequence and the majority are large co-factor containing proteins.
There is now considerable evidence that Tat systems can transport such globular
proteins in a fully folded state. The minimal components required for transport in
E.coli are TatA, TatB and TatC; these three integral membrane proteins are thought
to form an active translocon. In Bacillus subtilis only TatA and TatC subunits are
present, with TatA acting in a bifunctional manner to replace TatB. Little structural
information is known about these multimeric integral membrane protein complexes
due to the inherent difficulty in purifying them and their compositional variability.
Complexes formed by B. subtilis TatAd and TatAyCy and E. coli TatE were
investigated by single-particle EM analysis. An image processing protocol was
developed to analyse and separate out individual Tat complexes based on their size.
Using this method 3D electron density maps were generated of TatAd and TatE,
which appear as small, ring-shaped complexes. Unlike E. coli TatA complexes, that
have been shown to vary widely in size, those observed here appear small and
homogeneous. These data conflict with the widely accepted ‘size-fitting pore’ model
of Tat mediated translocation and rather support the alternative transient coalescent
model.
Additionally the first structural characterisation of a TatA-type mutant protein was
performed revealing a dramatic polymerisation phenotype and indicating a primary
role for the N-terminus in forming protein-protein interactions
Assembly and mechanism of bacterial twin-arginine translocation systems
No full textThe bacterial twin-arginine translocation (Tat) pathway is able to export pre-folded
cofactor containing proteins across the cytoplasmic membrane. Tat substrates bear
cleavable N-terminal signal peptides that are characterized by the presence of a
critical and highly conserved twin-arginine motif which lends the Tat pathway its
name.
In Escherichia coli and many other Gram-negative bacteria, three integral membrane
proteins: TatA, TatB and TatC are essential for Tat-dependent translocation. In
contrast Bacillus subtilis possesses a simpler TatAC system which lacks the TatB
component. In E. coli the TatA protein assembles into homo-oligomeric complexes
that vary considerably in size. The TatA proteins found in B. subtilis do not exhibit
the same degree of heterogeneity and this suggested mechanistic differences between
the Tat pathways of Gram-negative and Gram-positive bacteria. How the Tat system
works is still poorly understood, and the work presented in this thesis sought to gain
insights into the assembly and mechanism of E. coli and B. subtilis Tat pathways.
This work focused on the study of two previously uncharacterized components: the
E. coli TatA paralog TatE subunit and B. subtilis TatAc subunit.
In this thesis the purification and characterization of E. coli TatE complexes is
reported. Using analytical gel filtration chromatography, blue-native gelelectrophoresis
(BN-PAGE) and single-particle analysis of purified TatE complexes,
it was found that the TatE complexes are more discrete than the highly
heterogeneous TatA complexes. This finding, together with the ability of TatE to
support the translocation of the 90-kDa TorA protein, suggested alternative
translocation models in which single TatE complexes do not contribute the bulk of
the translocation channel, similar to the B. subtilis model.
In addition, co-purification and BN-PAGE experiments demonstrated for the first
time that TatE interacts with TatA to form TatAE mixed complexes in the
membrane, and reveals a completely novel form of Tat complex that might be
functionally significant.
A soluble population of TatE was also identified in E. coli cell extracts, and phase
separation experiments using Triton X-114 suggested it may be mis-localized.
In a separate set of studies, the ability of the B. subtilis TatAc protein to form active
translocases in combination with the B. subtilis TatCd or TatCy proteins was
investigated for the first time. The TatAcCd and TatAcCy mixed translocases were
able to translocate several E. coli Tat substrates including, TorA, AmiA and AmiC.
Finally BN-PAGE and gel filtration chromatography showed that the TatAcCd and
TatAcCy complexes were significantly smaller than the previously described E. coli
TatABC substrate-binding complex
Ubiquitin-proteasome system and mitochondrial respiration alterations andoxidative stress induction in human glioblastoma cells: role of the M2 muscarinicreceptors
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