[3H]MK-801 Binding to N-Methyl-d-Aspartate Receptors Solubilized from Rat Brain: Effects of Glycine Site Ligands, Polyamines, Ifenprodil, and Desipramine

The N-methyl-D-aspartate (NMDA) receptor is thought to contain several distinct binding sites that can regulate channel opening. In the present experiments, the effects of ligands for these sites have been examined on [3H]MK-801 binding to a soluble receptor preparation, which had been passed down a gel filtration column to reduce the levels of endogenous small-molecular-weight substances. Glycine site agonists, partial agonists, and antagonists gave effects similar to those observed in membranes [EC50 values (in microM): glycine, 0.31; D-serine, 0.20; D-cycloserine, 1.46; (+)-HA-966, 4.06; and 7-chlorokynurenic acid, 1.81]. Spermine and spermidine enhanced [3H]MK-801 binding to the soluble receptor preparation (EC50, 4.3 and 20.1 microM, respectively), whereas putrescine and cadaverine gave small degrees of inhibitions. When spermine and spermidine were tested under conditions where [3H]MK-801 binding approached equilibrium, their ability to enhance [3H]MK-801 binding was much reduced, a result suggesting that the polyamines increase the rate to equilibrium. Putrescine antagonised the effects of spermine. Ifenprodil reduced [3H]MK-801 binding under both equilibrium and nonequilibrium conditions, although the high-affinity component of inhibition described in membranes was not observed. Ifenprodil antagonised spermine effects in an apparently noncompetitive manner. Desipramine was able to give total inhibition of specific [3H]MK-801 binding under nonequilibrium conditions with an IC50 of 4 microM, and this value was unaltered when [3H]MK-801 binding was allowed to reach equilibrium. These results suggest that the sites mediating the effects of glycine and its analogues, polyamines and desipramine are integral components of the NMDA receptor protein.     

Mutagenicity of a unique apurinic/apyrimidinic site in mammalian cells.

Abasic sites are common DNA lesions produced either spontaneously or as a consequence of the action of some genotoxic agent. The mutagenic properties of a unique abasic site replicated in mammalian cells have been studied using a shuttle vector. A plasmid, able to replicate both in mammalian cells and in bacteria, carrying a unique abasic site chemically synthesized has been constructed. After replication in mammalian cells, plasmid DNA was recovered and used to transform bacteria. Mutants were screened without selection pressure by differential hybridization with a labelled oligonucleotide and their DNA was sequenced. A mutation frequency ranging from 1% to 3% was found, depending on the base originally inserted during the vector construction, opposite the abasic site. All the sequenced mutants correspond to single base-pair substitutions targeted at the abasic site. We observed a deficit in guanine incorporation opposite the abasic site, while the three other bases were incorporated with a similar efficiency. The mutational potency of abasic sites was observed without any voluntary preconditioning treatment of mammalian cells in order to induce "SOS" like conditions.     

An antigen located in the kinetochore region in metaphase and on polar microtubule ends in the midbody region in anaphase,characterised using a monoclonal antibody

We describe a new component of the kinetochore region of Chinese hamster ovary cells, which was characterised using a monoclonal antibody (mAb). This antigen was localised on the kinetochore regions of purified metaphase chromosomes, but in anaphase it was instead located on the polar microtubules in the midbody region, where they terminate in the stembody. It was not detectable in prophase or interphase cells by immunofluorescence, but was present in the interphase nucleus as shown by immunoblotting after SDS-polyacrylamide gel electrophoresis. The mAb recognised two polypeptides of M_r 140 000 and 155 000. The localisation of this antigen in metaphase on the kinetochore region, where the plus ends of the kinetochore microtubules are temporarily stabilised when they attach, and later in the stembody and midbody where the plus ends of the polar microtubules are stabilised in anaphase and telophase, suggests that it could play a role in stabilising the plus ends of microtubules and thus in the control of microtubule dynamics during mitosis.     

Key Features of Intertidal Food Webs That Support Migratory Shorebirds

The migratory shorebirds of the East Atlantic flyway land in huge numbers during a migratory stopover or wintering on the French Atlantic coast. The Brouage bare mudflat (Marennes-Oléron Bay, NE Atlantic) is one of the major stopover sites in France. The particular structure and function of a food web affects the efficiency of carbon transfer. The structure and functioning of the Brouage food web is crucial for the conservation of species landing within this area because it provides sufficient food, which allows shorebirds to reach the north of Europe where they nest. The aim of this study was to describe and understand which food web characteristics support nutritional needs of birds. Two food-web models were constructed, based on in situ measurements that were made in February 2008 (the presence of birds) and July 2008 (absence of birds). To complete the models, allometric relationships and additional data from the literature were used. The missing flow values of the food web models were estimated by Monte Carlo Markov Chain – Linear Inverse Modelling. The flow solutions obtained were used to calculate the ecological network analysis indices, which estimate the emergent properties of the functioning of a food-web.The total activities of the Brouage ecosystem in February and July are significantly different. The specialisation of the trophic links within the ecosystem does not appear to differ between the two models. In spite of a large export of carbon from the primary producer and detritus in winter, the higher recycling leads to a similar retention of carbon for the two seasons. It can be concluded that in February, the higher activity of the ecosystem coupled with a higher cycling and a mean internal organization, ensure the sufficient feeding of the migratory shorebirds.     

Shrub clearing adversely affects the abundance of Ixodes ricinus ticks

In order to get a better understanding of the importance of vertical forest structure as a component of Ixodes ricinus tick habitat, an experiment was set up in a coniferous forest on sandy soils in northern Belgium. Ticks were sampled in six control and six treatment plots on various sampling occasions in 2008–2010. In the course of the study period, a moderate thinning was carried out in all plots and shrub clearing was performed in the treatment plots. Thinning had no effect on tick abundance, while shrub clearing had an adverse affect on the abundance of all three life stages (larva, nymph, adult) up to 2 years post-clearing. Our findings are especially relevant in the light of the ongoing efforts to improve vertical forest structure in Belgium and many other parts of Europe, which might create suitable habitats for ticks and change the epidemiology of tick-borne diseases. Also, our results indicate that shrub clearing could be applied as a tick control measure in recreational areas where there is a high degree of human-tick contact.     

Mutations in BICD2, which Encodes a Golgin and Important Motor Adaptor,Cause Congenital Autosomal-Dominant Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is a heterogeneous group of neuromuscular disorders caused by degeneration of lower motor neurons. Although functional loss of SMN1 is associated with autosomal-recessive childhood SMA, the genetic cause for most families affected by dominantly inherited SMA is unknown. Here, we identified pathogenic variants in bicaudal D homolog 2 (Drosophila) (BICD2) in three families afflicted with autosomal-dominant SMA. Affected individuals displayed congenital slowly progressive muscle weakness mainly of the lower limbs and congenital contractures. In a large Dutch family, linkage analysis identified a 9q22.3 locus in which exome sequencing uncovered c.320C>T (p.Ser107Leu) in BICD2. Sequencing of 23 additional families affected by dominant SMA led to the identification of pathogenic variants in one family from Canada (c.2108C>T [p.Thr703Met]) and one from the Netherlands (c.563A>C [p.Asn188Thr]). BICD2 is a golgin and motor-adaptor protein involved in Golgi dynamics and vesicular and mRNA transport. Transient transfection of HeLa cells with all three mutant BICD2 cDNAs caused massive Golgi fragmentation. This observation was even more prominent in primary fibroblasts from an individual harboring c.2108C>T (p.Thr703Met) (affecting the C-terminal coiled-coil domain) and slightly less evident in individuals with c.563A>C (p.Asn188Thr) (affecting the N-terminal coiled-coil domain). Furthermore, BICD2 levels were reduced in affected individuals and trapped within the fragmented Golgi. Previous studies have shown that Drosophila mutant BicD causes reduced larvae locomotion by impaired clathrin-mediated synaptic endocytosis in neuromuscular junctions. These data emphasize the relevance of BICD2 in synaptic-vesicle recycling and support the conclusion that BICD2 mutations cause congenital slowly progressive dominant SMA.     

Claudin 7 is reduced in uterine epithelial cells during early pregnancy in the rat

The non-receptive uterine luminal epithelium forms an intact polarised epithelial barrier that is refractory to blastocyst invasion. During implantation, organised dismantling of this barrier leads to a receptive state promoting blastocyst attachment. Claudins are tight junction proteins that increase in the uterine epithelium at the time of implantation. Claudin 7 is a member of this family but demonstrates a basolateral localisation pattern that is distinct from other claudins. The present study investigated the localisation, abundance and hormonal regulation of claudin 7 to elucidate a role for the protein during implantation. The results showed that claudin 7 demonstrates a distinct basal and lateral localisation in the uterine luminal and glandular epithelium throughout early pregnancy. On day 1, claudin 7 is abundantly present in response to ovarian estrogen. At the time of implantation, claudin 7 decreases in abundance. This decrease is not dependent on blastocyst presence, as shown by results in pseudopregnant animals. We propose that claudin 7 mediates intercellular adhesions in the uterine epithelium and also may be responsible for stabilising adhesion proteins at the basolateral cell surface. Thus, claudin 7 may function under the maintenance of the uterine luminal epithelial barrier, in the non-receptive state preventing implantation from occurring.     

SNPflow: A Lightweight Application for the Processing,Storing and Automatic Quality Checking of Genotyping Assays

Single nucleotide polymorphisms (SNPs) play a prominent role in modern genetics. Current genotyping technologies such as Sequenom iPLEX, ABI TaqMan and KBioscience KASPar made the genotyping of huge SNP sets in large populations straightforward and allow the generation of hundreds of thousands of genotypes even in medium sized labs. While data generation is straightforward, the subsequent data conversion, storage and quality control steps are time-consuming, error-prone and require extensive bioinformatic support. In order to ease this tedious process, we developed SNPflow. SNPflow is a lightweight, intuitive and easily deployable application, which processes genotype data from Sequenom MassARRAY (iPLEX) and ABI 7900HT (TaqMan, KASPar) systems and is extendible to other genotyping methods as well. SNPflow automatically converts the raw output files to ready-to-use genotype lists, calculates all standard quality control values such as call rate, expected and real amount of replicates, minor allele frequency, absolute number of discordant replicates, discordance rate and the p-value of the HWE test, checks the plausibility of the observed genotype frequencies by comparing them to HapMap/1000-Genomes, provides a module for the processing of SNPs, which allow sex determination for DNA quality control purposes and, finally, stores all data in a relational database. SNPflow runs on all common operating systems and comes as both stand-alone version and multi-user version for laboratory-wide use. The software, a user manual, screenshots and a screencast illustrating the main features are available at http://genepi-snpflow.i-med.ac.at.     

Familial Creutzfeldt-Jakob Disease: Case report and role of genetic counseling in post mortem testing

Here we present a case of an asymptomatic 53-year-old woman who sought genetic testing for Familial Creutzfeldt-Jakob Disease (fCJD) after learning that her mother had fCJD. The patient's mother had a sudden onset of memory problems and rapidly deteriorating mental faculties in her late 70s, which led to difficulties ambulating, progressive non-fluent aphasia, dysphagia and death within ～1 y of symptom onset. The cause of death was reported as “rapid onset dementia.” The patient's family, unhappy with the vague diagnosis, researched prion disorders online and aggressively pursued causation and submitted frozen brain tissue from the mother to the National Prion Disease Surveillance Center, where testing revealed a previously described 5-octapeptide repeat insertion (5-OPRI) in the prion protein gene (PRNP) that is known to cause fCJD. The family had additional questions about the implications of this result and thus independently sought out genetic counseling.

?While rare, fCJD is likely underdiagnosed due to clinical heterogeneity, rapid onset, early non-specific symptomatology, and overlap in the differential diagnosis of Alzheimer disease and Lewy body dementias. When fCJD is identified, a multidisciplinary approach to return of results that includes the affected patient's provider, genetics professionals, and mental health professionals is key to the care of the family. We present an example case which discusses the psychosocial issues encountered and the role of genetic counseling in presymptomatic testing for incurable neurodegenerative conditions. Ordering physicians should be aware of the basic issues surrounding presymptomatic genetic testing and identify local genetic counseling resources for their patients.