Molekulare Mechanismen der cAMP-vermittelten Signaltransduktion

Zusammenfassung - Der sekundäre Botenstoff zyklisches Adenosinmonophosphat (cAMP) reguliert viele fundamentale zelluläre Prozesse wie Zellproliferation, Differenzierung, Energiemetabolismus und Genexpression. In eukaryotischen Zellen vermittelt die cAMP-abhängige Proteinkinase (PKA) die meisten biologischen Funktionen von cAMP. Die PKA besteht aus jeweils zwei regulatorischen (R) und katalytischen (C) Untereinheiten, die zusammen einen inaktiven Holoenzymkomplex bilden, der durch cAMP aktiviert wird. In dieser Arbeit wurde die Bindung von cAMP und cAMP-Analoga an die R Untereinheit der PKA unter funktionellen und mechanistischen Aspekten untersucht. Eine neue, auf Fluoreszenzpolarisation basierende Methode wurde entwickelt, um die Affinität von cAMP-Analoga in einem homogenen Ansatz schnell, reproduzierbar und nicht radioaktiv zu quantifizieren. Zur detaillierten Untersuchung des Bindungsmechanismus von cAMP und cAMP Analoga (Agonisten und Antagonisten) wurden thermodynamische Studien im direkten Vergleich mittels isothermaler Titrationskalorimetrie und kinetischen Analysen (Oberflächenplasmonresonanz, SPR) durchgeführt, wodurch thermodynamische Signaturen für das Bindungsverhalten der Nukleotide an die R Untereinheit der PKA erhalten werden konnten. Durch Interaktionsstudien an mutagenisierten R Untereinheiten wurde der intramolekulare Aktivierungsmechanismus der PKA in Bezug auf cAMP-Bindung, Holoenzymkomplex-Formierung und -Aktivierung untersucht. Die dabei erhaltenen Ergebnisse wurden mit zwei Modellen der cAMP-induzierten Konformationsänderung verglichen, und ein Aktivierungsmechanismus postuliert, der auf konservierten hydrophoben Aminosäuren basiert. Für in vivo Untersuchungen wurden zusammen mit Kooperationspartnern membranpermeable, fluoreszierende cAMP Analoga entwickelt, die Einblicke in die Dynamik der cAMP-Verteilung in Zellen erlauben. Neu entwickelte, Festphasen gebundene cAMP-Analoga (Agonisten und Antagonisten) wurden in einem (sub)proteomischen Ansatz dazu genutzt, natürliche Komplexe der R Untereinheit und des PKA-Holoenzyms aus Zelllysaten zu isolieren und zu identifizieren. Diese Untersuchungen fließen letztlich in einem systembiologischen Ansatz zusammen, der neue Einblicke in die vielschichtigen cAMP gesteuerten Netzwerke und Regulationsprozesse erlaubt.

Uso del test de cuantificación sensitiva para el estudio de la neuropatía de fibra pequeña

Introducción La mutación genética Val30Met de la proteína transtiretina (TTR) es causante de la polineuropatía amiloidótica familiar, comprometiendo en fases iniciales las fibras nerviosas pequeñas (mielinizadas Aδ y amielínicas tipo C), involucradas en funciones autonómicas, nocicepción, percepción térmica y sudoración. Los métodos neurofisiológicos convencionales, no logran detectar dichas anormalidades, retardando el inicio de tratamientos específicos para la enfermedad. Metodología El objetivo principal fue evaluar el test de cuantificación sensitiva (QST) como método de detección temprana de anormalidades de fibra pequeña, en individuos Val30Met, seguidos en el Hospital Universitario Santa María, Lisboa. Se clasificaron los pacientes en 3 grupos, según sintomatología y examen neurológico. Se analizaron los umbrales para percepción de frío, dolor con el calor y vibración en los grupos, en correlación con controles sanos. Resultados 18 registros de controles sanos y 33 de individuos con la mutación, divididos en asintomáticos (24,2%), sintomáticos con examen neurológico normal (42,4%) y sintomáticos con examen neurológico anormal (33,3%). No se encontraron diferencias entre los pacientes asintomáticos y los controles. Los umbrales para frío (p=0,042) y en el dolor intermedio con el calor (HP 5) (p=0,007) se encuentran elevados en individuos Val30Met sintomáticos con examen normal. En los pacientes sintomáticos con alteraciones al examen, también se presentaron alteraciones en el intervalo entre el inicio y el dolor intermedio con el calor (HP 5-0,5) (p=0,009). Discusión Los umbrales de frío y de percepción de dolor con el calor, permiten detectar anormalidades en personas con la mutación TTR Val30Met, sintomáticos, incluyendo aquellos sin cambios objetivos al examen neurológico.

Puesta en marcha de una biblioteca escolar

Se describe la puesta en marcha de la biblioteca del IES San Mart??n (Talayuela, C??ceres). Se detalla c??mo se form?? la colecci??n y se acondicionaron las instalaciones y se exponen las actividades llevadas a cabo para su dinamizaci??n

Crystal structure of the complementary quadruplex formed by d(GCATGCT) at atomic resolution

Here we report the crystal structure of the DNA heptanucleotide sequence d(GCATGCT) determined to a resolution of 1.1 Angstrom. The sequence folds into a complementary loop structure generating several unusual base pairings and is stabilised through cobalt hexammine and highly defined water sites. The single stranded loop is bound together through the G(N2)-C(O2) intra-strand H-bonds for the available G/C residues, which form further Watson-Crick pairings to a complementary sequence, through 2-fold symmetry, generating a pair of non-planar quadruplexes at the heart of the structure. Further, four adenine residues stack in pairs at one end, H-bonding through their N7-N6 positions, and are additionally stabilised through two highly conserved water positions at the structural terminus. This conformation is achieved through the rotation of the central thymine base at the pinnacle of the loop structure, where it stacks with an adjacent thymine residue within the lattice. The crystal packing yields two halved biological units, each related across a 2-fold symmetry axis spanning a cobalt hexammine residue between them, which stabilises the quadruplex structure through H-bonds to the phosphate oxygens and localised hydration.

Severe acute respiratory syndrome coronavirus nonstructural proteins 3, 4, and 6 induce double-membrane vesicles

Coronaviruses (CoV), like other positive-stranded RNA viruses, redirect and rearrange host cell membranes for use as part of the viral genome replication and transcription machinery. Specifically, coronaviruses induce the formation of double-membrane vesicles in infected cells. Although these double-membrane vesicles have been well characterized, the mechanism behind their formation remains unclear, including which viral proteins are responsible. Here, we use transfection of plasmid constructs encoding full-length versions of the three transmembrane-containing nonstructural proteins (nsps) of the severe acute respiratory syndrome (SARS) coronavirus to examine the ability of each to induce double-membrane vesicles in tissue culture. nsp3 has membrane disordering and proliferation ability, both in its full-length form and in a C-terminal-truncated form. nsp3 and nsp4 working together have the ability to pair membranes. nsp6 has membrane proliferation ability as well, inducing perinuclear vesicles localized around the microtubule organizing center. Together, nsp3, nsp4, and nsp6 have the ability to induce double-membrane vesicles that are similar to those observed in SARS coronavirus-infected cells. This activity appears to require the full-length form of nsp3 for action, as double-membrane vesicles were not seen in cells coexpressing the C-terminal truncation nsp3 with nsp4 and nsp6. IMPORTANCE Although the majority of infections caused by coronaviruses in humans are relatively mild, the SARS outbreak of 2002 to 2003 and the emergence of the human coronavirus Middle Eastern respiratory syndrome (MERS-CoV) in 2012 highlight the ability of these viruses to cause severe pathology and fatality. Insight into the molecular biology of how coronaviruses take over the host cell is critical for a full understanding of any known and possible future outbreaks caused by these viruses. Additionally, since membrane rearrangement is a tactic used by all known positive-sense single-stranded RNA viruses, this work adds to that body of knowledge and may prove beneficial in the development of future therapies not only for human coronavirus infections but for other pathogens as well.

Characterization of two novel polyfunctional mastoparan peptides from the venom of the social wasp Polybia paulista

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Normal Forms for Codimension One Planar Piecewise Smooth Vector Fields

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Tsukamoto s Theorem in Characteristic two

In this paper it is proved that hermitian forms over quaternion division algebras over local fields of characteristic two are classified by their dimension and discriminant.

Pathology of sarcoptic mange in red foxes (Vulpes vulpes): macroscopic and histologic characterization of three disease stages

Sarcoptic mange is a highly contagious skin disease that can have a devastating impact on affected wild mammal populations. There are notable variations in the clinical and pathologic picture of sarcoptic mange among species and among conspecifics. However, the origin of these variations is unclear. We propose a classification scheme for skin lesions associated with Sarcoptes scabiei infestation to provide a basis for a subsequent risk factor analysis. We conducted a case-control study focused on macroscopic and histologic examination of the skin, using 279 red foxes (Vulpes vulpes) found dead or shot in Switzerland between November 2004 and February 2006. All animals were submitted to gross necropsy following a detailed protocol. Selection criteria for cases (n=147) vs. controls (n=111) were the presence or absence of mange-like lesions, mite detection by isolation or histologic examination, and serologic testing for S. scabiei antibodies. Characteristic features of mange lesions were scored macroscopically in all foxes and histologically in 67 cases and 15 controls. We classified skin lesions and associated necropsy findings into three types of mange: A) early stage (n=45): focal-extensive skin lesions, thin crusts, mild to moderate alopecia, few mites, numerous eosinophils, and mild lymph node enlargement; B) hyperkeratotic, fatal form (n=86): generalized skin lesions, thick crusts with or without alopecia, foul odor, abundance of mites, numerous bacteria and yeasts, numerous lymphocytes and mast cells, severe lymph node enlargement, and emaciation; C) alopecic, healing form (n=16): focal lesions, no crusts, severe alopecia, hyperpigmentation and lichenification, absence of mites, mixed cell infiltration, and rare mild lymph node enlargement. We hypothesize that after stage A, the animal either enters stage B and dies, or stage C and survives, depending on largely unknown extrinsic or intrinsic factors affecting the host ability to control mite infestation.

Mg/Ca and d18O measurements of planktonic foraminifera from Sargasso Sea

Paired Mg/Ca and d18O measurements on planktonic foraminiferal species (G. ruber white, G. ruber pink, G. sacculifer, G. conglobatus, G. aequilateralis, O. universa, N. dutertrei, P. obliquiloculata, G. inflata, G. truncatulinoides, G. hirsuta, and G. crassaformis) from a 6-year sediment trap time series in the Sargasso Sea were used to define the sensitivity of foraminiferal Mg/Ca to calcification temperature. Habitat depths and calcification temperatures were estimated from comparison of d18O of foraminifera with equilibrium calcite, based on historical temperature and salinity data. When considered together, Mg/Ca (mmol/mol) of all species, except two, show a significant (r = 0.93) relationship with temperature (T °C) of the form Mg/Ca = 0.38 (±0.02) exp 0.090 (±0.003)T, equivalent to a 9.0 ± 0.3% change in Mg/Ca for a 1°C change in temperature. Small differences exist in calibrations between species and between different size fractions of the same species. O. universa and G. aequilateralis have higher Mg/Ca than other species, and in general, data can be best described with the same temperature sensitivity for all species and pre-exponential constants in the sequence O. universa > G. aequilateralis = G. bulloides > G. ruber = G. sacculifer = other species. This approach gives an accuracy of ±1.2°C in the estimation of calcification temperature. The 9% sensitivity to temperature is similar to published studies from culture and core top calibrations, but differences exist from some literature values of pre-exponential constants. Different cleaning methodologies and artefacts of core top dissolution are probably implicated, and perhaps environmental factors yet understood. Planktonic foraminiferal Mg/Ca temperature estimates can be used for reconstructing surface temperatures and mixed and thermocline temperatures (using G. ruber pink, G. ruber white, G. sacculifer, N. dutertrei, P. obliquiloculata, etc.). The existence of a single Mg thermometry equation is valuable for extinct species, although use of species-specific equations will, where statistically significant, provide more accurate evaluation of Mg/Ca paleotemperature.

Regulation of the human p21/WAF1/Cip1 promoter in hepatic cells by functional interactions between Sp1 and Smad family members

The cell cycle inhibitor p21/WAF1/Cip1 is expressed in many cell types and is regulated by p53-dependent and p53-independent mechanisms. p21 is an important regulator of hepatocyte cell cycle, differentiation, and liver development, but little is known about the regulation of its synthesis in hepatocytes. We report herein that the p21 gene is constitutively expressed in human hepatoma HepG2 cells. Deletion analysis of the p21 promoter showed that it contains a distal (positions −2,300/−210) and a proximal (positions −124 to −61) region that act synergistically to achieve high levels of constitutive expression. The proximal region that consists of multiple Sp1 binding sites is essential for constitutive p21 promoter activity in hepatocytes. This region also mediates the transcriptional activation of the p21 promoter by members of the Smad family of proteins, which play important role in the transduction of extracellular signals such as transforming growth factor β, activin, etc. Constitutive expression of p21 was severely reduced by a C-terminally truncated form of Smad4 that was shown previously to block signaling through Smads. Smad3/4 and to a much lesser extent Smad2/4 caused high levels of transcriptional activation of the p21 promoter. Transactivation was compromised by N- or C-terminally truncated forms of Smad3. By using Gal4-Sp1 fusion proteins, we show that Smad proteins can activate gene transcription via functional interactions with the ubiquitous factor Sp1. These data demonstrate that Smad proteins and Sp1 participate in the constitutive or inducible expression of the p21 gene in hepatic cells.

Myc represses the p21(WAF1/CIP1) promoter and interacts with Sp1/Sp3

The cyclin-dependent kinase inhibitor p21(WAF1/CIP1) inhibits proliferation both in vitro and in vivo, and overexpression of p21 in normal and tumor cell lines results in cell cycle arrest. In contrast, ectopic expression of Myc alleviates G1 cell cycle arrest. Recent studies showed that Myc can repress p21 transcription, thereby overriding a p21-mediated cell cycle checkpoint. We found that activation of a Myc-estrogen receptor fusion protein by 4-hydroxytamoxifen in mouse cells resulted in suppression of endogenous p21 transcription. This effect was observed in the absence of de novo protein synthesis and was independent of histone deacetylase activity. In transient transfection studies, Myc effectively repressed p21 promoter constructs containing only 119 bp of sequence upstream of the transcription start site. This region contains multiple Sp1-binding sites and a potential initiator element, but no canonical Myc DNA-binding sites. Deletion of the potential initiator element does not affect repression of the p21 promoter by c-Myc. Coimmunoprecipitation and glutathione S-transferase pull-down experiments demonstrate that c-Myc may form complexes with Sp1/Sp3. We found that the central region of c-Myc interacts with the zinc finger domain of Sp1. Because Sp1 is required for p21 transcription, it is possible that Myc may down-regulate p21 transcription, at least in part, by sequestering Sp1. Repression of the p21 promoter may contribute to the ability of c-Myc to promote cell proliferation.

Zeta functions and Eisenstein series on classical groups

We construct an Euler product from the Hecke eigenvalues of an automorphic form on a classical group and prove its analytic continuation to the whole complex plane when the group is a unitary group over a CM field and the eigenform is holomorphic. We also prove analytic continuation of an Eisenstein series on another unitary group, containing the group just mentioned defined with such an eigenform. As an application of our methods, we prove an explicit class number formula for a totally definite hermitian form over a CM field.

Subnuclear targeting of Runx/Cbfa/AML factors is essential for tissue-specific differentiation during embryonic development

Runx (Cbfa/AML) transcription factors are critical for tissue-specific gene expression. A unique targeting signal in the C terminus directs Runx factors to discrete foci within the nucleus. Using Runx2/CBFA1/AML3 and its essential role in osteogenesis as a model, we investigated the fundamental importance of fidelity of subnuclear localization for tissue differentiating activity by deleting the intranuclear targeting signal via homologous recombination. Mice homozygous for the deletion (Runx2ΔC) do not form bone due to maturational arrest of osteoblasts. Heterozygotes do not develop clavicles, but are otherwise normal. These phenotypes are indistinguishable from those of the homozygous and heterozygous null mutants, indicating that the intranuclear targeting signal is a critical determinant for function. The expressed truncated Runx2ΔC protein enters the nucleus and retains normal DNA binding activity, but shows complete loss of intranuclear targeting. These results demonstrate that the multifunctional N-terminal region of the Runx2 protein is not sufficient for biological activity. We conclude that subnuclear localization of Runx factors in specific foci together with associated regulatory functions is essential for control of Runx-dependent genes involved in tissue differentiation during embryonic development.

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