A topological model for the haemolysin translocator protein HlyD

Summary A topological model for HlyD is proposed that is based on results obtained with gene fusions of lacZ and phoA to hlyD. Active H1yD-LacZ fusion proteins were only generated when lacZ was fused to hlyD. within the first 180 by (60 amino acids). H1yD-PhoA proteins exhibiting alkaline phosphatase (AP) activity were obtained when phoA was inserted into hlyD. between nucleotides 262 (behind amino acid position 87) and 1405 (behind amino acid position 468, only 10 amino acids away from the C-terminus of HlyD Active insertions of phoA into the middle region of hlyD. were not observed on in vivo transposition but such fusions exhibiting AP activity could be constructed by in vitro techniques. A fusion protein that carried the PhoA part close to the C-terminal end of HlyD proved to be the most stable HlyD-PhoA fusion protein. In contrast to the other, rather unstable, HlyD-PhoA⁺ fusions, no proteolytic degradation product of this HlyD-PhoA protein was observed and nearly all the alkaline phosphatase activity was membrane bound. Protease accessibility and cell fractionation experiments indicated that the alkaline phosphatase moiety of this fusion protein was located in the periplasm as for all other HlyD-PhoA⁺ proteins. These data and computer-assisted predictions suggest a topological model for HlyD with the N-terminal 60 amino acids located in the cytoplasm, a single transmembrane segment from amino acids 60 to 80 and a large periplasmic region extending from amino acid 80 to the C-terminus. Neither the HlyD fusion proteins obtained nor a mutant HlyD protein that had lost the last 10 amino acids from the C-terminus of HlyD exhibited translocator activity for HlyA or other reporter proteins carrying the HlyA signal sequence. The C-terminal 10 amino acids of HlyD showed significant similarity with the corresponding sequences of other HlyD-related proteins involved in protein secretion.     

Non-synonymous gene polymorphisms in the secretory signal peptide of human TGF-β1 affect cellular synthesis but not secretion of TGF-β1

We have demonstrated that gene polymorphisms within the N-terminal leader sequence of TGF-β1 contribute to the outcome of hepatic fibrogenesis. In addition, the polymorphism at codon 25 affects TGF-β1 production in peripheral blood leukocytes. Therefore, it is general assumed that these polymorphisms influence cellular secretion of this cytokine. In the present study, we analysed if this widespread hypothesis is true. We cloned FLAG-tagged CMV-driven human full-length TGF-β1 expression constructs of the different allelic variations (i.e. 10Leu/25Arg, 10Pro/25Pro and 10Pro/25Arg) and transfected them into the immortal hepatic stellate cell line LX-2 and Chinese Hamster Ovary cells. Surprisingly, the allelic variants carrying a proline either in codon 10 or 25 showed overall reduced expression as assessed by Western blot and quantitative ELISA. We conclude that the allelic variations within the signal sequence influence the expression and not secretion of TGF-β1. Detailed RNA structure prediction analysis further suggests that the individual variants form different secondary structures.     

An extraction method for nematodes in decomposition studies using the minicontainer-method

The minicontainer-method is a new method developed to study biological processes related to soil litter decomposition. An adaptation of the classical Baermann-funnel technique is described which can be used, in association with the minicontainer method, to investigate the role of Nematoda in litter decomposition. The use of the extraction method is illustrated in a study of the effects of different tillage systems on the decomposition of rye straw and on the nematode density in minicontainers with different mesh sizes of 20 µm, 500 µm and 2 mm. Three tilled plots (conventional deep plough, cultivator and two-layer plough) and an untilled control were compared after periods of 4 weeks and 38 weeks. On both sample dates there were significant main effects of treatment and mesh size on the nematode density, and additionally, after 38 weeks significant treatment x soil depth interactions. After 4 weeks, there were significant main effects of treatment and soil depth on the decomposition, but no mesh size effects, whereas after 38 weeks, all experimental factors had a significant effect on the decomposition of the straw. Due to the small volume of litter substrate used in the minicontainer method, the efficiency of nematode extraction is high and the lack of oxygen in the minicontainers presents no serious problem during the extraction process. The method also allows the simultaneous extraction of a large number of samples within a short period of time. Our results indicate that the method is suitable to study the microdistribution of nematode activity within the soil profile and improves the possible applications of the minicontainer-method.     

Roles of cAMP and cAMP-dependent protein kinase in the progression of prostate cancer: Cross-talk with the androgen receptor

Prostate carcinomas are among the most frequently diagnosed and death causing cancers affecting males in the developed world. It has become clear that the molecular mechanisms that drive the differentiation of normal prostate cells towards neoplasia involve multiple signal transduction cascades that often overlap and interact. A critical mediator of cellular proliferation and differentiation in various cells (and cancers) is the cAMP-dependent protein kinase, also known as protein kinase A (PKA), and its activating secondary messenger, cAMP. PKA and cAMP have been shown to play critical roles in prostate carcinogenesis and are the subject of this review. In particular we will focus on the cross-talk between PKA/cAMP signaling and that of the androgen receptor.     

Helical packaging of semiflexible polymers in bacteriophages

We investigate multilayered helical packaging of double-stranded DNA, or of a general polymer chain with persistence length l_b, into an ideal, inert cylindrical container, reaching densities slightly below close packaging. We calculate the free energy as a function of the packaged length, based on the energies for bending, twisting, the suffered entropy loss, and the electrostatic energy in a Debye–Hückel model. In the absence of charges on the packaged polymer, a critical packaging force can be determined, similar to the mechanism involved in DNA unzipping models. When charges are taken into consideration, in the final packaging state the charges which are chemically distant become geometrically close, and therefore a steep rise is seen in the free energy. We argue that due to the extremely ordered and almost closely packaged final state the actual packaging geometry does not influence the behaviour of the free energy, pointing towards a certain universality of this state of the polymer. Our findings are compared to a recent simulations study, showing that the model is sensitive to the screening length.     

Porous polymers for fixed bed adsorption of aroma compounds in fermentation processes

Summary Deionized water as well as simulated and genuine fermentation media, which contained varying concentrations of benzaldehyde and 4-decanolide, were used to investigate the applicability of selected styrene-divinylbenzene resins for low pressure downflow adsorption of aroma compounds in a fixed bed. The effects of flow rate and matrix on the respective breakthrough curve slopes were examined using the LUB-and the MTZ-model.     

Self-esteem in adolescent patients with attention-deficit/hyperactivity disorder during open-label atomoxetine treatment: psychometric evaluation of the Rosenberg Self-Esteem Scale and clinical findings

To report on (1) psychometric properties of the Rosenberg Self-Esteem Scale (SES) studied in adolescents with ADHD, (2) correlations of SES with ADHD scale scores, and (3) change in patient-reported self-esteem with atomoxetine treatment. ADHD patients (12–17 years), treated in an open-label study for 24 weeks. Secondary analyses on ADHD symptoms (assessed with ADHD-RS, CGI, GIPD scales) and self-esteem (SES) were performed. One hundred and fifty-nine patients were treated. A dichotomous structure of the SES could be confirmed. Reliability and internal consistency were moderate to excellent. Highest coefficients were found for the correlation between SES and GIPD scores. Self-esteem significantly increased over time, accompanied by an improvement of ADHD symptoms and related perceived difficulties. The Rosenberg SES was shown to be internally consistent, reliable, and sensitive to treatment-related changes of self-esteem. According to these findings, self-esteem may be an important individual patient outcome beyond the core symptoms of ADHD.     

Heterotopic Cervical Heart Transplantation in Mice

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies this model by avoiding challenging suture anastomoses of small vessels thereby reducing warm ischemia time. In comparison to abdominal graft implantation the cervical model is less invasive and the implanted graft is easily accessible for further follow-up examinations. Anastomoses are performed by pulling the ascending aorta of the graft over the cuff with the recipient’s common carotid artery and by pulling the main pulmonary artery over the cuff with the external jugular vein. Selection of appropriate cuff size and complete mobilization of the vessels are important for successful revascularization. Ischemia-reperfusion (I/R) injury can be minimized by perfusing the graft with a cardioplegic solution and by hypothermia. In this article, we provide technical details for a simplified and improved cuff technique, which should allow surgeons with basic microsurgical skills to perform the procedure with a high success rate.