Ontogeny of PAC1-R and VPAC1-R in the frog, Rana esculenta

The distribution of pituitary adenylate cyclase-activating polypeptide (PACAP) and PACAP receptors in the brain of amphibians has been previously described. In the present study, we have investigated the ontogeny of the selective PACAP receptor, PAC1-R, and the PACAP-vasoactive intestinal polypeptide (VIP) mutual receptor, VPAC1-R, in frog embryos by whole-mount in situ hybridization histochemistry. At stage 20, expression of PAC1-R and/or VPAC1-R mRNAs was detected in the brain, the auditory vesicles, the external gills, the buds of the lateral lines and the coelomatic cavity. At stage 25, PAC1-R and/or VPAC1-R mRNAs were observed in the buds of the orbital lateral line, the pancreas and heart. At stage 30, PAC1-R and VPAC1-R mRNAs were widely distributed in the telencephalon and diencephalon as well as in the bud of the lateral line, the heart and the pancreas. The anatomical distribution of PAC1-R and VPAC1-R mRNAs, although similar, did not totally overlap, indicating that PACAP and VIP may exert differential effects in frog during development.     

Generation of Leishmania mutants lacking antibiotic resistance genes using a versatile hit-and-run targeting strategy

The development of a method to create defined mutants of Leishmania parasites lacking foreign genes conferring resistance to antibiotics has both experimental and practical applications. Mutants deficient in specific virulence genes have potential as attenuated live vaccines, but these can only be of clinical relevance if the antibiotic resistance genes used for selection of the mutants are subsequently removed. In addition, the limited number of antibiotic resistance genes that can be used for genetic manipulation of Leishmania means that a system for recycling them for subsequent use would be highly beneficial when multiple genetic modifications are wanted. In the method we report here, a cassette carrying in tandem the hygromycin resistance gene as a positive marker and thymidine kinase gene as a negative marker is first integrated into the locus of interest and then replaced by a null targeting fragment containing no exogenous DNA. The application of this hit-and-run strategy for removal of one allele of the CPB cysteine peptidase gene array of Leishmania infantum is described.     

Induction of crassulacean acid metabolism in Mesembryanthemum crystallinum increases reproductive success under conditions of drought and salinity stress

Summary Mesembryanthemum crystallinum L., an inducible crassulacean acid metabolism (CAM) plant, was grown for approximately 5 weeks following germination in well-watered, non-saline soil in a controlled-environment chamber. During this time, plants were characterized by C₃ photosynthetic carbon metabolism. After the initial 5 weeks, CAM was induced by a combination of high soil salinity and reduced soil water content. One group of plants was allowed to engage in CAM by being continuously exposed to normal CO₂-containing air (about 350–400 ppm). A second group of plants was deprived of ambient CO₂ each night (12 h dark period) until completion of their life cycle, thereby minimizing potential carbon gain via dark CO₂ fixation. The capacity to express CAM under conditions of drought and salinity stress markedly improved reproductive success: plants kept in normal CO₂-containing air produced about 10 times more seeds than plants kept in CO₂-free air during dark periods. Seeds from plants deprived of ambient CO₂ overnight had more negative ¹³C values than seeds from plants kept in normal air.     

Serumprotein polymorphisms in Iran

Summary The results of a population genetic investigation on Iranians are given and compared to the results obtained on other populations from Southwestern and Southern Asia. Our total material from Iran comprises n=1020 nonrelated male and female individuals of different age. The following serum groups have been typed: Hp, Gc, Gm, and Inv. In general there exist no remarkable age or sex differences in the distribution of phenotypes and alleles (the only exception: sex differences in the distribution of the Gm (7)-phenotype). The regional distribution of phenotypes and alleles yield no marked differences, too, apart from the Invphenotypes, however. For the total material of Iran the following alleles frequencies could be calculated: Hp¹=0.3045, Hp²=0.6595, Gc²=0.3405; Gm¹=0.1780, Gm^1,2=0.0537, Gm^1,5=0.0632, Gm⁵=0.7051. The Gm (7)-phenotype turned out to be 36.6%; the Inv (1)-phenotype amounts to 25.6%. Comparing with other populations, especially Pakistani and Indian samples, some heterogeneity in the distribution of phenotypes and alleles within Southwestern and Southern Asia was to be demonstrated. Some distributional trends of alleles frequencies shall be mentioned here: the increase of Hp², Gc¹, and Gm¹ alleles from West towards East, and in the opposite direction the decrease of Hp¹, Gc², and Gm⁵ alleles. Selective acting forces are supposed to be most important factors for this. D77     

Spontaneous polar anterior subcapsular lenticular opacity in Sprague-Dawley rats

A spontaneous focal polar anterior subcapsular lenticular opacity characterized by focal epithelial proliferation was found in Charles River Sprague-Dawley rats from various breeding facilities around the world (France, Japan, and the United States). The incidence of this change slightly increased with age up to a maximal incidence of 9.8% in 28- to 35-week-old male rats (French source). Over that period, there was little change in the size of the opacity; however some rats that were examined over longer periods (more than 2 years of age) developed secondary anterior cortical changes, and rarely, histologic findings of pigmentation and/or mineralization. The lenticular change was present throughout the life of the animals and had no sex predilection; mode of inheritance was not investigated. Due to its small size, this lens opacity is more easily identified by use of slit lamp biomicroscopy than by use of indirect ophthalmoscopy, and serial sections of the eye aid in locating it for histologic evaluation.     

A Standard Operating Procedure (SOP) for the preparation of intra- and extracellular proteins of Clostridium acetobutylicum for proteome analysis

We report on the development of a Standard Operating Procedure (SOP) for extraction and handling of intra- and extracellular protein fractions of Clostridium acetobutylicum ATCC 824 for reproducible high quality two-dimensional gel electrophoresis (2-DE) analyses. Standardized cells from a phosphate-limited chemostat were used to evaluate different protein preparation methods. For the preparation of the secretome, a dialysis/ultrafiltration procedure resulted in higher protein yields and proved to be more reliable compared to different precipitation methods using TCA, DOC-TCA, acetone, and PEG 6000. Sonication was found to be the most efficient method among different tested techniques of cell disruption for the analysis of the intracellular proteome. Furthermore, the effect of protease inhibitors and sample storage conditions were tested for both intra- and extracellular protein samples. Significant changes in the protein pattern were observed depending on the addition of protease inhibitors. 2-DE gels with a pH gradient from 4 to 7 prepared according to the developed SOP contained at least 736 intracellular and 324 extracellular protein spots.     

Membrane perturbation by an external electric field: a mechanism to permit molecular uptake

Electropermeabilisation is a well established physical method, based on the application of electric pulses, which induces the transient permeabilisation of the cell membrane. External molecules, otherwise nonpermeant, can enter the cell. Electropermeabilisation is now in use for the delivery of a large variety of molecules, as drugs and nucleic acids. Therefore, the method has great potential in the fields of cancer treatment and gene therapy. However many open questions about the underlying physical mechanisms involved remain to be answered or fully elucidated. In particular, the induced changes by the effects of the applied field on the membrane structure are still far from being fully understood. The present review focuses on questions related to the current theories, i.e. the basic physical processes responsible for the electropermeabilisation of lipid membranes. It also addresses recent findings using molecular dynamics simulations as well as experimental studies of the effect of the field on membrane components. Presented at the joint biannual meeting of the SFB-GEIMM-GRIP, Anglet France, 14–19 October, 2006.     

Glycan chains modulate prion protein binding to immobilized metal ions

PrP(c) is the normal isoform of the prion protein which can be converted into PrP(Sc), the pathology-associated conformer in prion diseases. It contains two N-linked glycan chains attached to the C-proximal globular domain. While the biological functions of PrP(c) are still unknown, its ability to bind Cu(2+) is well documented. The main Cu(2+)-binding sites are located in the N-proximal, unstructured region of the molecule. Here we report that PrP(c) glycans influence the capacity of PrP(c) from sheep brain or cultured Rov cells to bind IMAC columns loaded with Cu(2+) or Co(2+). Using different anti-PrP antibodies and PrP(c) glycosylation mutants, we show that the full length non-glycosylated form of PrP(c) has a higher binding efficiency for column-bound Cu(2+) and Co(2+) than the corresponding glycosylated form. Our findings raise the possibility that the accessibility of the PrP(c) metal ion-binding sites might be controlled by the glycan chains.     

Deciphering the molecular effects of romidepsin on germ cell tumours: DHRS2 is involved in cell cycle arrest but not apoptosis or induction of romidepsin effectors

Testicular germ cell tumours (GCTs) mostly affect young men at age 17‐40. Although high cure rates can be achieved by orchiectomy and chemotherapy, GCTs can still be a lethal threat to young patients with metastases or therapy resistance. Thus, alternative treatment options are needed. Based on studies utilising GCT cell lines, the histone deacetylase inhibitor romidepsin is a promising therapeutic option, showing high toxicity at very low doses towards cisplatin‐resistant GCT cells, but not fibroblasts or Sertoli cells. In this study, we extended our analysis of the molecular effects of romidepsin to deepen our understanding of the underlying mechanisms. Patients will benefit from these analyses, since detailed knowledge of the romidepsin effects allows for a better risk and side‐effect assessment. We screened for changes in histone acetylation of specific lysine residues and analysed changes in the DNA methylation landscape after romidepsin treatment of the GCT cell lines TCam‐2, 2102EP, NCCIT and JAR, while human fibroblasts were used as controls. In addition, we focused on the role of the dehydrogenase/reductase DHRS2, which was strongly up‐regulated in romidepsin treated cells, by generating DHRS2‐deficient TCam‐2 cells using CRISPR/Cas9 gene editing. We show that DHRS2 is dispensable for up‐regulation of romidepsin effectors (GADD45B, DUSP1, ZFP36, ATF3, FOS, CDKN1A, ID2) but contributes to induction of cell cycle arrest. Finally, we show that a combinatory treatment of romidepsin plus the gluccocorticoid dexamethasone further boosts expression of the romidepsin effectors and reduces viability of GCT cells more strongly than under single agent treatment. Thus, romidepsin and dexamethasone might represent a new combinatorial approach for treatment of GCT.