TGF-beta isoform signaling regulates secondary transition and mesenchymal-induced endocrine development in the embryonic mouse pancreas

Transforming growth factor-beta (TGF-beta) superfamily signaling has been implicated in many developmental processes, including pancreatic development. Previous studies are conflicting with regard to an exact role for TGF-beta signaling in various aspects of pancreatic organogenesis. Here we have investigated the role of TGF-beta isoform signaling in embryonic pancreas differentiation and lineage selection. The TGF-beta isoform receptors (RI, RII and ALK1) were localized mainly to both the pancreatic epithelium and mesenchyme at early stages of development, but then with increasing age localized to the pancreatic islets and ducts. To determine the specific role of TGF-beta isoforms, we functionally inactivated TGF-beta signaling at different points in the signaling cascade. Disruption of TGF-beta signaling at the receptor level using mice overexpressing the dominant-negative TGF-beta type II receptor showed an increase in endocrine precursors and proliferating endocrine cells, with an abnormal accumulation of endocrine cells around the developing ducts of mid-late stage embryonic pancreas. This pattern suggested that TGF-beta isoform signaling may suppress the origination of secondary transition endocrine cells from the ducts. Secondly, TGF-beta isoform ligand inhibition with neutralizing antibody in pancreatic organ culture also led to an increase in the number of endocrine-positive cells. Thirdly, hybrid mix-and-match in vitro recombinations of transgenic pancreatic mesenchyme and wild-type epithelium also led to increased endocrine cell differentiation, but with different patterns depending on the directionality of the epithelial-mesenchymal signaling. Together these results suggest that TGF-beta signaling is important for restraining the growth and differentiation of pancreatic epithelial cells, particularly away from the endocrine lineage. Inhibition of TGF-beta signaling in the embryonic period may thus allow pancreatic epithelial cells to progress towards the endocrine lineage unchecked, particularly as part of the secondary transition of pancreatic endocrine cell development. TGF-beta RII in the ducts and islets may normally serve to downregulate the production of beta cells from embryonic ducts.     

ATP-driven MalK dimer closure and reopening and conformational changes of the "EAA" motifs are crucial for function of the maltose ATP-binding cassette transporter (MalFGK2)

We have investigated conformational changes of the purified maltose ATP-binding cassette transporter (MalFGK(2)) upon binding of ATP. The transport complex is composed of a heterodimer of the hydrophobic subunits MalF and MalG constituting the translocation pore and of a homodimer of MalK, representing the ATP-hydrolyzing subunit. Substrate is delivered to the transporter in complex with periplasmic maltose-binding protein (MalE). Cross-linking experiments with a variant containing an A85C mutation within the Q-loop of each MalK monomer indicated an ATP-induced shortening of the distance between both monomers. Cross-linking caused a substantial inhibition of MalE-maltose-stimulated ATPase activity. We further demonstrated that a mutation affecting the "catalytic carboxylate" (E159Q) locks the MalK dimer in the closed state, whereas a transporter containing the "ABC signature" mutation Q140K permanently resides in the resting state. Cross-linking experiments with variants containing the A85C mutation combined with cysteine substitutions in the conserved EAA motifs of MalF and MalG, respectively, revealed close proximity of these residues in the resting state. The formation of a MalK-MalG heterodimer remained unchanged upon the addition of ATP, indicating that MalG-EAA moves along with MalK during dimer closure. In contrast, the yield of MalK-MalF dimers was substantially reduced. This might be taken as further evidence for asymmetric functions of both EAA motifs. Cross-linking also caused inhibition of ATPase activity, suggesting that transporter function requires conformational changes of both EAA motifs. Together, our data support ATP-driven MalK dimer closure and reopening as crucial steps in the translocation cycle of the intact maltose transporter and are discussed with respect to a current model.     

Tissue-specific autophagy alterations and increased tumorigenesis in mice deficient in Atg4C/autophagin-3

Atg4C/autophagin-3 is a member of a family of cysteine proteinases proposed to be involved in the processing and delipidation of the mammalian orthologues of yeast Atg8, an essential component of an ubiquitin-like modification system required for execution of autophagy. To date, the in vivo role of the different members of this family of proteinases remains unclear. To gain further insights into the functional relevance of Atg4 orthologues, we have generated mutant mice deficient in Atg4C/autophagin-3. These mice are viable and fertile and do not display any obvious abnormalities, indicating that they are able to develop the autophagic response required during the early neonatal period. However, Atg4C-/--starved mice show a decreased autophagic activity in the diaphragm as assessed by immunoblotting studies and by fluorescence microscopic analysis of samples from Atg4C-/- GFP-LC3 transgenic mice. In addition, animals deficient in Atg4C show an increased susceptibility to develop fibrosarcomas induced by chemical carcinogens. Based on these results, we propose that Atg4C is not essential for autophagy development under normal conditions but is required for a proper autophagic response under stressful conditions such as prolonged starvation. We also propose that this enzyme could play an in vivo role in events associated with tumor progression.     

Relationships between body fat measured by DXA and subcutaneous adipose tissue thickness measured by Lipometer in adults

The aim of this study was to examine the relationships between body fat measured by DXA and subcutaneous adipose tissue layers (SAT-layers) measured by LIPOMETER in adult males (n=28) and females (n=53). Body height and mass were measured and BMI was calculated (kg/m2). Measurements of the thicknesses of SAT-layers by LIPOMETER were performed at 15 original body sites. Body composition was measured using DXA. Total body fat % measured by DXA was highly dependent on the SAT-layers in the upper back and inner thigh in males (87.1%, R(2)x100) and the lateral chest, biceps, and calf in females (78.5%, R(2)x100). There were gender differences in trunk fat mass and right hand and leg fat mass calculation using specific SAT-layers. In conclusion, our results indicate that there are close relationships between SAT-layers and body fat measured by DXA. However, there are big differences between genders.     

Evaluation of risk profiles by subcutaneous adipose tissue topography in obese juveniles

Objective: To compare subcutaneous adipose tissue topography (SAT‐top) in obese juveniles with age‐matched normal‐weight controls. Research Methods and Procedures: The optical device LIPOMETER (European Patent EP 0516251) enables the non‐invasive, rapid, safe, and precise measurement of the thickness of subcutaneous adipose tissue. Fifteen defined body sites (1 = neck to 15 = calf) characterize the individual SAT‐top like an individual fingerprint. SAT‐top of 1351 juveniles (obese: 42 boys, 59 girls, normal weight: 680 boys, 570 girls) from 7 to 19 years of age were measured. For visual comparison, the 15‐dimensional SAT‐top information was condensed by factor analysis into a two‐dimensional factor plot. Results: Both female and male obese juveniles had markedly increased adipose tissue layers at 7 = upper abdomen, 8 = lower abdomen, 5 = front chest, and 6 = lateral chest. The pubertal changes of body shape and fat distribution of the normal‐weight boys and girls (boys show thinner adipose tissue layers on their legs, whereas girls had thicker adipose tissue layers at the extremities) were not seen in the obese group. Independently of age and sex, all of the obese juveniles showed a similar, more android body fat distribution with increased trunk fat. Discussion: SAT‐top of the obese juveniles is similar to that of patients with type 2 diabetes, polycystic ovary syndrome, and coronary heart disease. Patients with these metabolic disorders and obese juveniles are located in the factor plot in the same area. This body shape may indicate a risk profile for developing polycystic ovary syndrome (women), type 2 diabetes, and early atherosclerosis (both sexes).     

Breast MRI in nonpalpable breast lesions: a randomized trial with diagnostic and therapeutic outcome – MONET – study

Background

In orthodontic treatment, anchorage control is a fundamental aspect. Usually conventional mechanism for orthodontic anchorage control can be either extraoral or intraoral that is headgear or intermaxillary elastics. Their use are combined with various side effects such as tipping of occlusal plane or undesirable movements of teeth. Especially in cases, where key-teeth are missing, conventional anchorage defined as tooth-borne anchorage will meet limitations. Therefore, the use of endosseous implants for anchorage purposes are increasingly used to achieve positional stability and maximum anchorage.

Methods/Design

The intended study is designed as a prospective, multicenter randomized controlled trial (RCT), comparing and contrasting the effect of early loading of palatal implant therapy versus implant loading after 12 weeks post implantation using the new ortho-implant type II anchor system device (Orthosystem Straumann, Basel, Switzerland). 124 participants, mainly adult males or females, whose diagnoses require temporary stationary implant-based anchorage treatment will be randomized 1:1 to one of two treatment groups: group 1 will receive a loading of implant standard therapy after a healing period of 12 week (gold standard), whereas group 2 will receive an early loading of orthodontic implants within 1 week after implant insertion. Participants will be at least followed for 12 months after implant placement. The primary endpoint is to investigate the behavior of early loaded palatal implants in order to find out if shorter healing periods might be justified to accelerate active orthodontic treatment. Secondary outcomes will focus e.g. on achievement of orthodontic treatment goals and quantity of direct implant-bone interface of removed bone specimens. As tertiary objective, a histologic and microtomography evaluation of all retrieved implants will be performed to obtain data on the performance of the SLA surface in human bone evaluation of all retrieved implants. Additionally, resonance frequency analysis (RFA, Osstell? mentor) will be used at different times for clinically monitoring the implant stability and for histological comparison in order to measure the reliability of the resonance frequency measuring device.

Trial registration

Current Controlled Trials ISRCTN97142521.     

Molecular and genomic studies of IMMP2L and mutation screening in autism and Tourette syndrome

We recently reported the disruption of the inner mitochondrial membrane peptidase 2-like (IMMP2L) gene by a chromosomal breakpoint in a patient with Gilles de la Tourette syndrome (GTS). In the present study we sought to identify genetic variation in IMMP2L, which, through alteration of protein function or level of expression might contribute to the manifestation of GTS. We screened 39 GTS patients, and, due to the localization of IMMP2L in the critical region for the autistic disorder (AD) locus on chromosome 7q (AUTS1), 95 multiplex AD families; however, no coding mutations were found in either GTS or AD patients. In addition, no parental-specific expression of IMMP2L was detected in somatic cell hybrids containing human chromosome 7 and human cell lines carrying a maternal uniparental disomy for chromosome 7 (mUPD7). Despite the fact that no deleterious mutations in IMMPL2 (other than the inverted duplication identified previously) were identified in either GTS or AD, this gene cannot be excluded as a possible rare cause of either disorder.     

A tandem affinity purification-based technology platform to study the cell cycle interactome in Arabidopsis thaliana

Defining protein complexes is critical to virtually all aspects of cell biology because many cellular processes are regulated by stable protein complexes, and their identification often provides insights into their function. We describe the development and application of a high throughput tandem affinity purification/mass spectrometry platform for cell suspension cultures to analyze cell cycle-related protein complexes in Arabidopsis thaliana. Elucidation of this protein-protein interaction network is essential to fully understand the functional differences between the highly redundant cyclin-dependent kinase/cyclin modules, which are generally accepted to play a central role in cell cycle control, in all eukaryotes. Cell suspension cultures were chosen because they provide an unlimited supply of protein extracts of actively dividing and undifferentiated cells, which is crucial for a systematic study of the cell cycle interactome in the absence of plant development. Here we report the mapping of a protein interaction network around six known core cell cycle proteins by an integrated approach comprising generic Gateway-based vectors with high cloning flexibility, the fast generation of transgenic suspension cultures, tandem affinity purification adapted for plant cells, matrix-assisted laser desorption ionization tandem mass spectrometry, data analysis, and functional assays. We identified 28 new molecular associations and confirmed 14 previously described interactions. This systemic approach provides new insights into the basic cell cycle control mechanisms and is generally applicable to other pathways in plants.     

Effect of sterol structure on ordered membrane domain (raft) stability in symmetric and asymmetric vesicles

Sterol structure influences liquid ordered domains in membranes, and the dependence of biological functions on sterol structure can help identify processes dependent on ordered domains. In this study we compared the effect of sterol structure on ordered domain formation in symmetric vesicles composed of mixtures of sphingomyelin, 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol, and in asymmetric vesicles in which sphingomyelin was introduced into the outer leaflet of vesicles composed of DOPC and cholesterol. In most cases, sterol behavior was similar in symmetric and asymmetric vesicles, with ordered domains most strongly stabilized by 7-dehydrocholesterol (7DHC) and cholesterol, stabilized to a moderate degree by lanosterol, epicholesterol and desmosterol, and very little if at all by 4-cholesten-3-one. However, in asymmetric vesicles desmosterol stabilized ordered domain almost as well as cholesterol, and to a much greater degree than epicholesterol, so that the ability to support ordered domains decreased in the order 7-DHC > cholesterol > desmosterol > lanosterol > epicholesterol > 4-cholesten-3-one. This contrasts with values for intermediate stabilizing sterols in symmetric vesicles in which the ranking was cholesterol > lanosterol ~ desmosterol ~ epicholesterol or prior studies in which the ranking was cholesterol ~ epicholesterol > lanosterol ~ desmosterol. The reasons for these differences are discussed. Based on these results, we re-evaluated our prior studies in cells and conclude that endocytosis levels and bacterial uptake are even more closely correlated with the ability of sterols to form ordered domains than previously thought, and do not necessarily require that a sterol have a 3β-OH group.     

Biologie, ökologie und brutpflege von coelotes terrestris (wider) (araneae,agelenidae) teil I. biologie und ökologie

Zusammenfassung Coelotes terrestris (Wider) hat seinen Verbreitungsschwerpunkt in den geschlossenen Laub- und Nadelwäldern besserer Bonität auf lehmigen Böden und kommt in erster Linie in Berggegenden vor. Von dort strahlt sie aber weit auf die Sandböden der Ebene aus und kompensiert dabei die Feuchtigkeitsdifferenz zwischen Lehm- und Sandboden durch ihren Aufenthalt in Gebieten mit hohem Grundwasserstand. Den wechselnden Feuchtigkeitsverhältnissen entsprechend ist ihr Vorkommen in Verbreitungsrandgebieten inselartig. Wenn sie stellenweise auch in Grenzgebieten möglicher Besiedlung, wie sie scheinbar trockene, dürftige Föhrenbestände darstellen, eine überraschend hohe Populationsdichte erreicht, ist dies nicht auf ein Optimum an Faktorenkonstellation schlechthin, sondern auf die Unmöglichkeit einer Ausbreitung in angrenzende Trockengebiete zurückzuführen. Coelotes ist eine Bodenspinne. Auf lehmigen Böden lebt sie in Laubstreu, in Bodenspalten unter den flach ausgebreiteten Wurzeln von Fichten, auf Sandboden in hochwüchsigem Moos (Hypneen, nicht Sphagnum), vor allem aber in der Rohhumus- und Streuschicht, die sockelartig den Fuß von Föhrenstämmen umgibt, und überall gerne unter großen, tiefliegenden Steinen. Bewohner von Kiefernstämmen legen ihre Netze mit Vorliebe zwischen Borke und umgebender Rohhumus-bzw. Streuschicht an. Um einen Kiefernstamm von nur 16 cm Durchmesser wurden bis zu fünf bewohnte Netze gefunden. Eine Prävalenz von Kiefernborke war im Wahlversuch nachweisbar, desgleichen eine solche von Rohhumus vor ebenso feuchtem Lehm- oder Sandboden verschiedener Körnigkeit. Dank seiner beträchtlichen Wasserkapazität und guten Wärme-Isolation bietet der Rohhumus diesen Spinnen auch in Trockengebieten eine Existenzmöglichkeit. Resistenzversuche ergaben, daß Coelotes zwar auf die Dauer eine weitgehende Wasserdampfsättigung der Luft benötigt, daß aber seine Widerstandsfähigkeit gegenüber einer relativen Luftfeuchtigkeit von 30–75% im Vergleich zu anderen feuchtigkeitssuchenden Bodentieren recht groß ist. Die Resistenz aller Jugendstadien ist höher als die der Erwachsenen. Während sich aber die Lebensdauer der noch im mütterlichen Netz verweilenden Kleinspinnen mit abnehmender Luftfeuchtigkeit merklich verkürzt, erweist sich die Widerstandsfähigkeit der halbwüchsigen Individuen, die überwintern, in allen Feuchtigkeitsbereichen als annähernd gleich hoch. Selbst in 75% relativer Luftfeuchtigkeit blieben adulte nur durchschnittlich 131 Std am Leben, bei 30% dagegen immerhin noch 40 Std. Jungspinnen hingegen halten in 30% relativer Luftfeuchtigkeit 2–3mal so lange aus.Bei Stammbewohnern ließ sich eine Bevorzugung der Ostseite (nicht Osthälfte!) und noch deutlicher eine Meidung der West- und Nordwestseite der Stämme nachweisen, wodurch sie sich - ihrer Hemihygrophilie entsprechend - dem unmittelbaren Abfluß des Regenwassers von den Stämmen entziehen. Nächst der genauen Ostrichtung werden aber auch die Nord- und Südseiten der Stämme stark frequentiert, die Zwischenhimmelsrichtungen dagegen auffallend benachteiligt. Als Erklärung dafür wird angenommen, daß die Spinnen durch vermutliche polarisationsoptische Orientierung primär der Ostseite zustreben. Ist diese schon besetzt, rücken sie zur Wahrung eines angemessenen Sicherheitsabstandes vom Nachbarindividuum um rund 90° des Kreisbogens ab und treffen somit auf die Nord- oder Südseite. - Die Überwinterung der und halbwüchsigen Tiere erfolgt 10–15 cm von den Kiefernstämmen entfernt im Rohhumus und zwar nur etwa 5 cm unter der Bodenoberfläche. Dabei bevorzugen sie die Ostseite und relativ trockene Stellen. Seltener überwintern sie in ihren alten Wohnröhren, die sie dann oben verschließen.Habilitationsschrift