RepB proteins of the multipartite Rhizobium leguminosarum bv. trifolii genome discriminate between centromere‐like parS sequences for plasmid segregational stability

The plasmids of the Rhizobiaceae family members and other Alphaproteobacteria are usually large, low copy‐number and contain all elements necessary for active segregation and replication located in one operon comprising repABC genes. The genome of Rhizobium leguminosarum bv. trifolii TA1 (RtTA1) consists of a chromosome and four plasmids (pRleTA1a‐d) with repABC operons. In this work, centromere‐binding RepB proteins of four RtTA1 plasmids were studied. Stability assays of the truncated derivatives of repABC cassettes demonstrated that RepA, RepB proteins and parS‐like elements constituted plasmid partitioning systems, while RepC were sufficient for their replication. Individual RepB proteins bound specifically to centromere‐like parS elements of the parental plasmids, which was crucial step toward the proper segregation of plasmids into daughter cells. RtTA1 RepB proteins formed dimers and oligomers in the solution. The C‐terminal part of RepB was responsible for dimerization, while the domain engaged in parS binding was located in the middle of the protein. It was concluded that the specific interaction between individual RepB proteins and their target sequences together with the substantial diversity of the Rep proteins and parS originating from different plasmids strongly contributed to the coexistence of several plasmids equipped with similar repABC cassettes in the multipartite bacterial genome.     

How to Study Basement Membrane Stiffness as a Biophysical Trigger in Prostate Cancer and Other Age-related Pathologies or Metabolic Diseases

Here we describe a protocol that can be used to study the biophysical microenvironment related to increased thickness and stiffness of the basement membrane (BM) during age-related pathologies and metabolic disorders (e.g. cancer, diabetes, microvascular disease, retinopathy, nephropathy and neuropathy). The premise of the model is non-enzymatic crosslinking of reconstituted BM (rBM) matrix by treatment with glycolaldehyde (GLA) to promote advanced glycation endproduct (AGE) generation via the Maillard reaction. Examples of laboratory techniques that can be used to confirm AGE generation, non-enzymatic crosslinking and increased stiffness in GLA treated rBM are outlined. These include preparation of native rBM (treated with phosphate-buffered saline, PBS) and stiff rBM (treated with GLA) for determination of: its AGE content by photometric analysis and immunofluorescent microscopy, its non-enzymatic crosslinking by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) as well as confocal microscopy, and its increased stiffness using rheometry. The procedure described here can be used to increase the rigidity (elastic moduli, E) of rBM up to 3.2-fold, consistent with measurements made in healthy versus diseased human prostate tissue. To recreate the biophysical microenvironment associated with the aging and diseased prostate gland three prostate cell types were introduced on to native rBM and stiff rBM: RWPE-1, prostate epithelial cells (PECs) derived from a normal prostate gland; BPH-1, PECs derived from a prostate gland affected by benign prostatic hyperplasia (BPH); and PC3, metastatic cells derived from a secondary bone tumor originating from prostate cancer. Multiple parameters can be measured, including the size, shape and invasive characteristics of the 3D glandular acini formed by RWPE-1 and BPH-1 on native versus stiff rBM, and average cell length, migratory velocity and persistence of cell movement of 3D spheroids formed by PC3 cells under the same conditions. Cell signaling pathways and the subcellular localization of proteins can also be assessed.     

Morphology and odor sensitivity of regenerated snail tentacles

This study examined certain structural and functional aspects of the olfactory system in regenerated posterior tentacles of the terrestrial snail Achatina fulica. Regeneration of the epithelial sensory pad occurs with accurate size regulation. All five neuronal cell types which are normally revealed by horseradish peroxidase backfilling are also regenerated. The sensory cells attain normal numbers at about 20 weeks postlesion. The organization of neuronal elements within the tentacle is chaotic, however, at early stages of regeneration. Even later, the digitlike extensions of the ganglion, which are characteristic of intact tentacles, fail to appear. The recovery of odor sensitivity was evaluated using a tentacular olfactormeter and a behavioral assay that involved locomotor orientation towards the odor stimulus. Thresholds and concentration-dependent response rates were equivalent for regenerated and intact tentacles, tested in the same animals, at 10 weeks post-lesion.     

Glacial relics in the bryoflora of the highlands Českomoravská vrchovina (Bohemian-Moravian Highlands); Their habitat and cenotaxonomic value

The paper sums up the information which the author has so far collected about the habitats and phytocenotic conditions of the glacial relics ofScorpidium scorpioides (Hedw.) Limpr.,Calliergon trifarium (Web. etMohr.) Kindb.,Paludella squarrosa (Hedw.) Bred., andMeesia triquetra (Hook. etTayl.) Aongstr. in the Highlands ?eskomoravská vrchovina (Bohemian-Moravian Highlands) in Czechoslovakia. Simultaneously, it presents a survey of the localities in which the occurrence of these species has been ascertained up to now.