Recombinant prion protein induces rapid polarization and development of synapses in embryonic rat hippocampal neurons in vitro

While a beta-sheet-rich form of the prion protein (PrPSc) causes neurodegeneration, the biological activity of its precursor, the cellular prion protein (PrPC), has been elusive. We have studied the effect of purified recombinant prion protein (recPrP) on rat fetal hippocampal neurons in culture. Overnight exposure to Syrian hamster or mouse recPrP, folded into an alpha-helical-rich conformation similar to that of PrPC, resulted in a 1.9-fold increase in neurons with a differentiated axon, a 13.5-fold increase in neurons with differentiated dendrites, a fivefold increase in axon length, and the formation of extensive neuronal circuitry. Formation of synaptic-like contacts was increased by a factor of 4.6 after exposure to recPrP for 7 days. Neither the N-terminal nor C-terminal domains of recPrP nor the PrP paralogue doppel (Dpl) enhanced the polarization of neurons. Inhibitors of protein kinase C (PKC) and of Src kinases, including p59Fyn, blocked the effect of recPrP on axon elongation, while inhibitors of phosphatidylinositol 3-kinase showed a partial inhibition, suggesting that signaling cascades involving these kinases are candidates for transduction of recPrP-mediated signals. The results predict that full-length PrPC functions as a growth factor involved in development of neuronal polarity.     

Association of neuregulin 1 with schizophrenia confirmed in a Scottish population

Recently, we identified neuregulin 1 (NRG1) as a susceptibility gene for schizophrenia in the Icelandic population, by a combined linkage and association approach. Here, we report the first study evaluating the relevance of NRG1 to schizophrenia in a population outside Iceland. Markers representing a core at-risk haplotype found in Icelanders at the 5' end of the NRG1 gene were genotyped in 609 unrelated Scottish patients and 618 unrelated Scottish control individuals. This haplotype consisted of five SNP markers and two microsatellites, which all appear to be in strong linkage disequilibrium. For the Scottish patients and control subjects, haplotype frequencies were estimated by maximum likelihood, using the expectation-maximization algorithm. The frequency of the seven-marker haplotype among the Scottish patients was significantly greater than that among the control subjects (10.2% vs. 5.9%, P=.00031). The estimated risk ratio was 1.8, which is in keeping with our report of unrelated Icelandic patients (2.1). Three of the seven markers in the haplotype gave single-point P values ranging from .000064 to .0021 for the allele contributing to the at-risk haplotype. This direct replication of haplotype association in a second population further implicates NRG1 as a factor that contributes to the etiology of schizophrenia.     

Acetylcholinesterase inhibitory activity of lycopodane-type alkaloids from the Icelandic Lycopodium annotinum ssp. alpestre

The aim of this study was to investigate structures and acetylcholinesterase inhibitory activities of lycopodane-type alkaloids isolated from an Icelandic collection of Lycopodium annotinum ssp. alpestre. Ten alkaloids were isolated, including annotinine, annotine, lycodoline, lycoposerramine M, anhydrolycodoline, gnidioidine, lycofoline, lannotinidine D, and acrifoline, as well as a previously unknown N-oxide of annotine. ¹H and ¹³C NMR data of several of the alkaloids were provided for the first time. Solvent-dependent equilibrium constants between ketone and hemiketal form of acrifoline were determined. Conformation of acrifoline was characterized using NOESY spectroscopy and molecular modelling. The isolated alkaloids were evaluated for their in vitro inhibitory activity against acetylcholinesterase and butyrylcholinesterase. Ligand docking studies based on mutated 3D structure of Torpedo californica acetylcholinesterase provided rationale for low inhibitory activity of the isolated alkaloids as compared to huperzine A or B, which are potent acetylcholinesterase inhibitors belonging to the lycodine class. Based on the modelling studies the lycopodane-type alkaloids seem to fit well into the active site gorge of the enzyme but the position of their functional groups is not compatible with establishing strong hydrogen bonding interactions with the amino acid residues that line the binding site. The docking studies indicate possibilities of additional functionalization of the lycopodane skeleton to render potentially more active analogues.     

Preferential counterion binding to A-tract DNA oligomers

The free solution mobility of four 20 bp DNA oligomers, with and without A-tracts, has been measured by capillary electrophoresis in Tris-acetate buffer, to test the hypothesis that site-specific binding of monovalent counterions can occur in the narrow minor groove of A-tract DNAs. Preferential counterion binding has been proposed to cause A-tract bending because of asymmetric charge neutralization and collapse of the helix backbone toward the minor groove. Preferential counterion binding in A-tract DNAs should be manifested by a decrease in the electrophoretic mobility observed in free solution, compared to that of non-A-tract DNAs of the same size. Of the four sequences studied here, the slowest absolute mobility, indicative of the greatest counterion binding, was observed for a 20 bp oligomer containing two runs of A3T3 in phase with the helix repeat. A 20-mer containing phased CACA sequences migrated with the fastest mobility; 20-mers containing phased A5 tracts or phased runs of T3A3 migrated with intermediate mobilities. Very similar mobility differences were observed when 1-20 mM NaCl was added to the buffer. The results suggest that preferential counterion binding occurs in A-tract DNAs, especially those containing the AnTn sequence motif.     

Foraminiferan (Protozoa) epizoites on deep-water arcturids (Crustacea,Isopoda)

Foraminiferan (Protozoa) epizoites were examined on two deep-water isopods, Neastacilla sp. and Pleuroprion hystrix (Valvifera, Arcturidae), from the north Atlantic Ocean and the Nordic Seas. Most foraminiferans belonged to genus Cibicides and occurred on 27% of Neastacilla and 23% of Pleuroprion hystrix. The pattern of foraminiferan epibiosis was similar in both species, with a concentration on the dorsal body and on the posterior pereopods. The arcturids showed an increase in number of foraminiferans with increased body size, suggesting that available time span for settling on the younger stages (mancas) was relatively short. There was a trend towards larger numbers of foraminiferans occurring on larger arcturid species, suggesting that available space is the factor that determines the total number of foraminiferans for epibenthic arcturid isopod species.     

The association of a SNP upstream of INSIG2 with body mass index is reproduced in several but not all cohorts

A SNP upstream of the INSIG2 gene, rs7566605, was recently found to be associated with obesity as measured by body mass index (BMI) by Herbert and colleagues. The association between increased BMI and homozygosity for the minor allele was first observed in data from a genome-wide association scan of 86,604 SNPs in 923 related individuals from the Framingham Heart Study offspring cohort. The association was reproduced in four additional cohorts, but was not seen in a fifth cohort. To further assess the general reproducibility of this association, we genotyped rs7566605 in nine large cohorts from eight populations across multiple ethnicities (total n = 16,969). We tested this variant for association with BMI in each sample under a recessive model using family-based, population-based, and case-control designs. We observed a significant (p < 0.05) association in five cohorts but saw no association in three other cohorts. There was variability in the strength of association evidence across examination cycles in longitudinal data from unrelated individuals in the Framingham Heart Study Offspring cohort. A combined analysis revealed significant independent validation of this association in both unrelated (p = 0.046) and family-based (p = 0.004) samples. The estimated risk conferred by this allele is small, and could easily be masked by small sample size, population stratification, or other confounders. These validation studies suggest that the original association is less likely to be spurious, but the failure to observe an association in every data set suggests that the effect of SNP rs7566605 on BMI may be heterogeneous across population samples.     

An efficient and generic strategy for producing soluble human proteins and domains in E. coli by screening construct libraries

The implementation of generic and efficient technologies for the production of recombinant eukaryotic proteins remains an outstanding challenge in structural genomics programs. We have recently developed a new method for rapid identification of soluble protein expression in E. coli, the colony filtration blot (CoFi blot). In this study, the CoFi blot was used to screen libraries where the N-terminal translation start point was randomized. To investigate the efficiency of this strategy, we have attributed a large number of proteins to this process. In a set of 32 mammalian proteins, we were able to double the success rate (from 34 to 68%) of producing soluble and readily purifiable proteins in E. coli. Most of the selected constructs had their N-termini close to predicted domain borders and the method therefore provides a mean for experimental "domain foot printing." Surprisingly, for most of the targets, we also observed expressing constructs that were close to full-length. In summary this strategy constitutes a generic and efficient method for producing mammalian proteins for structural and functional studies.     

Retinal Oximetry with Scanning Laser Ophthalmoscope in Infants

Purpose

Dual wavelength retinal oximetry has been developed for adults, but is not available for infants. Retinal oximetry may provide insight into the pathophysiology of oxygen-mediated diseases like retinopathy of prematurity. More insight in the oxygen metabolism of the retina in infants may provide valuable clues for better understanding and subsequent prevention or treatment of the disease. The measurements of oxygen saturation are obtained with two fundus images simultaneously captured in two different wavelengths of light. The comparison in light absorption of oxygenated and deoxygenated hemoglobin can be used to estimate the oxygen saturation within the retinal vessels by means of a software algorithm. This study aims to make retinal oximetry available for neonates. The first step towards estimating retinal oxygen saturation is determining the optical density ratio. Therefore, the purpose of this study is to image healthy newborn infants with a scanning laser ophthalmoscope and determine the optical density ratio for retinal oximetry analysis.

Methods

Images of the retina of full-term healthy infants were obtained with an SLO, Optomap 200Tx (Optos), with two laser wavelengths (532nm and 633nm). The infant lay face down on the lower arm of the parent, while the parent supported the chest and chin with one hand, and stabilized the back with the other hand. No mydriatics or eyelid specula were used during this study. The images were analyzed with modified Oxymap Analyzer software for calculation of the Optical Density Ratio (ODR) and vessel width. The ODR is inversely and approximately linearly related to the oxygen saturation. Measurements were included from the superotemporal vessel pair. A paired t-test was used for statistical analysis.

Results

Fifty-nine infants, (58% female), were included with mean gestational age of 40 ± 1.3 weeks (mean ± SD) and mean post-natal age of 16 ± 4.8 days. A total of 28 images were selected for retinal oximetry analysis. The ODR was 0.256 ± 0.041 for the arterioles and 0.421 ± 0.089 for the venules (n = 28, p < 0.001). The measured vessel-width for the arterioles was 14.1 ± 2.7 pixels and for the venules 19.7 ± 3.7 pixels (n = 28, p < 0.001).

Conclusions

Retinal oximetry can be performed in newborn infants by combining an SLO and a dual-wavelength algorithm software. Sensitivity of the approach is indicated by the fact that the ODR measurements are significantly different between the arterioles and the venules. However, more variability in ODR is seen with the SLO approach in babies than is seen with conventional oximetry in adults. This approach is completely non-invasive, non-contact and even avoids the use of mydriatics or eyelid specula.